mecoprop-P (ISO); (R)-2-(4-chloro-2-methylphenoxy)propionic acid

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

One-Generation Reproductive Toxicity: Clode (2003)

Under the conditions of the study dietary administration of 1 200 ppm of the test material to adult male and female rats for 10 weeks prior to mating, 790 ppm to the female rats during the lactation period, and again giving 1 200 ppm to the F1 generation for 4 weeks, produced slight adult and pup toxicity in terms of lower body weight gains and food intakes. At these concentrations there were fewer implantation sites compared to control.

At 500 and 800 ppm (reduced to 300 and 530 ppm during lactation), there were marginal effects on adult and pup weight gain and food intake and slightly lower numbers of implantation sites compared to control.

It is considered that reduction of dietary concentration by approximately one third during the lactation period in a 2-generation study, would allow demonstration of a maternal MTD without inducing excessive effects in the offspring.

Two-Generation Reproductive Toxicity: Hellwig (1992)

Under the conditions of the study, the NOAEL for reproductive effects was 500 ppm (equivalent to ca. 40 mg/kg bw/day). The NOAEL for systemic toxicity of the test material was 100 ppm for F0 females, F1 and F2 pups, and 20 ppm for F0 males, for males and females of the P1 parental generation and the F1b pups.

Link to relevant study records

Reference

Reference 1

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 November 2002 to 29 April 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Glx/BRL/Han)BR

Details on species / strain selection:

The rat was selected as it is a readily available rodent species acceptable to the regulatory authorities and is recommended because of its reproductive characteristics and the quantity of published background data.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: At the start of treatment P males and females were approximately 7 to 9 weeks old.
- Weight at study initiation: At the start of treatment P males and females were within the weight range 227.4 to 280.3 g (males) and 166.0 to 210.6 g (females).
- Housing: Animals were housed in groups of up to four (pre-pairing and post-pairing), one female with one male (pairing). During the gestation and post-partum phases, females were housed individually in solid floored polycarbonate cages. The F1 generation animals were housed in groups of five in stainless steel wire mesh cages.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: All animals were given a clinical inspection for ill health on arrival. They were acclimatised for six days and not ten as stated in the protocol. A veterinary inspection was performed before the start of dosing to ensure their suitability for study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 40 to 70 % relative humidity. On several occasions the humidity was outside the protocol range, the highest recording being 74 %.
- Air changes (per hr): Air-conditioned to provide a minimum of 15 air changes/hour.
- Photoperiod (hrs dark / hrs light): Lighting was controlled automatically to give a cycle of 12 hours light (0600 to 1800) and 12 hours dark.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: Test diets of the test material in the vehicle were prepared weekly. The test material was weighed into pre-labelled beakers. The test material was transferred to a mortar containing a small quantity of diet. The test material was then ground down with the diet. The mixture was added directly to the diet in the mixing drum and mixed for 5 minutes.
- Storage temperature of food: The test diets were stored at room temperature (10 to 30 °C) in a sealed container.

Details on mating procedure:

- M/F ratio per cage: One male was housed with one female from the same treatment group.
- Length of cohabitation: After the growth/maturation phase one male was housed with one female from the same treatment group for up to 15 days.
- Proof of pregnancy: Mating was confirmed by the presence of a vaginal plug in situ or sperm in a vaginal washing. On confirmation of mating, vaginal washing was discontinued and the male was removed and rehoused. The day on which mating was confirmed was designated Day 0 of gestation.
- After successful mating each pregnant female was caged: Females were housed individually in solid floored polycarbonate cages.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Stability and homogeneity: Stability and homogeneity was performed for each batch of test material used on the study.
- Achieved concentrations: Samples were taken from the dietary formulations prepared for the first and last weeks of treatment of each generation and were analysed for achieved concentration.

Analysis of test diets in Weeks 1, 11, 17 and 22 of treatment showed that they were within the range 89 to 110 % of nominal, with the exception of one replicate sample of the Intermediate dose group formulation in Week 1, where a concentration of 117 % of nominal was obtained. Analysis of a third sample from this formulation gave a value of 101 % of nominal.

Duration of treatment / exposure:

Both sexes were treated for 10 weeks before pairing on a one to one basis within each group. The females continued with this treatment throughout gestation and then were allowed to litter and rear their offspring to weaning. During the lactation period, the females were given diets containing nominal concentrations of 0 (control), 300, 530 and 790 ppm test material. Males were treated until evaluation of the females had been completed. Ten animals of each sex were randomly selected from each group to form the filial (F1) generation. These animals received test material at the original concentrations for 4 weeks post-weaning.

Frequency of treatment:

Continuous in diet

Dose / conc.:

500 ppm

Remarks:

This dose level was fed during the pre-mating, gestation and post-weaning periods.

Dose / conc.:

800 ppm

Remarks:

This dose level was fed during the pre-mating, gestation and post-weaning periods.

Dose / conc.:

1 200 ppm

Remarks:

This dose level was fed during the pre-mating, gestation and post-weaning periods.

Dose / conc.:

300 ppm

Remarks:

During lactation, the inclusion levels were reduced to approximately 60 % for Group 2 (500 ppm group)

Dose / conc.:

530 ppm

Remarks:

During lactation, the inclusion levels were reduced to approximately 66 % for Group 3 (800 ppm group).

Dose / conc.:

790 ppm

Remarks:

During lactation, the inclusion levels were reduced to approximately 66 % for Group 4 (1 200 ppm group).

No. of animals per sex per dose:

12 per sex per dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Doses were selected by the Sponsor on the basis of previous studies.
In a 90-day rat study, a dose of 1 600 ppm test material in diet reduced growth in both male and female rats with some parallel changes in clinical chemistry (liver and kidney function tests) and haematology (red cell parameters). The target organ was established as the kidney which was increased in weight, both absolute and relative, compared to controls. Changes in clinical chemistry indicated that the liver may also be affected but histopathological change was not seen in either organ. In common with other phenoxy herbicides clinical signs of intoxication are seen only at very high (peri-lethal) doses. Thus the most sensitive index of effect for routine use is bodyweight. At 1 600 ppm the bodyweight effect was slightly greater than that required to establish MTD; a dose of 3 200 ppm was tolerated but effects were clearly in excess of the MTD. Thus, for the current study a high dose of 1 200 ppm was selected. It was anticipated that this would be sufficient to establish an MTD in the parental animals without excessive toxicity to the offspring.
In the 90-day study referred to above there were statistically significant but minor effects on weight gain in female (but not male) rats at a dietary inclusion rate of 800 ppm. The mid dose in the current study was set at this level in order to establish a fall-back position should the high dose prove excessive for the pups and to establish whether there are significant effects on pups at a dose where parental effects are minimal.
The low dose of 500 ppm was the high dose in the previous 2-generation study with the test material. There were only minor effects on the parents. It was anticipated that with the current design there would be a similar slight parental effect (although it may not have been evident with the small group size in this study), but by contrast with the previous study pup effects should be absent.
Compound consumption was calculated using the following formula:

Compound consumption (mg/kg/day) = (diet concentration (ppm) x food intake (g/day)) / mid-interval body weight (g)

- Rationale for animal assignment: The P animals were assigned to treatment groups during the acclimatisation period using a randomisation procedure based on stratified body weight. Allocation of the F1 generation was by random selection of 10 males and 10 females from the available litters.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined twice daily to detect any which were dead or moribund.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined at least once daily for signs of ill health or overt toxicity. Any abnormalities of appearance or behaviour or other signs of reaction to treatment or ill health were recorded and a detailed individual record was maintained of the clinical condition of each animal on the days of body weight recording.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded weekly for the males. For the females, individual body weights were recorded weekly during the growth/maturation phases until confirmation of mating, on Days 0, 7, 14 and 20 of gestation and on Days 1, 4, 7, 14 and 21 post-partum. Individual body weights were recorded weekly during the 4 weeks of maturation of the F1 generation.

FOOD CONSUMPTION AND COMPOUND INTAKE: YES
- The food consumed by each cage of animals was determined weekly during the pre-pairing periods (males and females).
Individual food intake was recorded daily and reported over Days 0 to 6, 7 to 13 and 14 to 19 of gestation (mated females) and over Days 1 to 3, 4 to 6, 7 to 13 and 14 to 20 post-partum (littering females) but reported on the body weight intervals.
Food intake for the F1 generation was measured over the same intervals as the body weights.

WATER CONSUMPTION AND COMPOUND INTAKE: No.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes.
- On Day 4 post-partum, litters were culled to a maximum of eight pups with an equal sex distribution where possible. Animals considered unlikely to survive to weaning were pre-selected for cull and a random selection procedure was used for additional pups.
- Weaning of the F1a offspring: On Day 21, 10 pups/sex were randomly selected from available litters for the F1 generation. Pups not selected were killed and necropsied.
- Maturation phase F1 generation: Selected F1 animals underwent at least 4 weeks of maturation, whilst receiving the control or test material formulations.


GROSS EXAMINATION OF DEAD PUPS:
- All pups found dead were examined macroscopically for structural or pathological changes. Culled pups were examined externally and discarded if normal. Abnormal pups were examined for structural or pathological changes.

Postmortem examinations (parental animals):

- Procedures for post-partum phase: All females were allowed to litter and rear offspring to weaning (Day 21 post-partum). The day pups were first observed was designated Day 0 post-partum. Abnormal signs of nesting, parturition or nursing were recorded.
- All animals were killed by an intraperitoneal injection of sodium pentobarbitone.
- All P animals were examined externally and discarded if found normal. Abnormal animals were examined for structural or pathological changes.
The uterus of all P females was stained in 10 % ammonium sulphide and the number of implantations recorded.

Postmortem examinations (offspring):

All F1 animals were examined externally and discarded if found normal. Abnormal animals were examined for structural or pathological changes.
All pups found dead were examined macroscopically for structural or pathological changes. Culled pups were examined externally and discarded if normal. Abnormal pups were examined for structural or pathological changes.

Statistics:

Body weight gains and food intake were analysed using one-way analysis of variance (ANOVA). Levene's test for equality of variances among the groups was performed. Where this showed no evidence of heterogeneity (P ≥ 0.01), pairwise comparisons with control were made using Dunnett's test. A linear contrast was performed to determine whether there was a relationship between increasing dose and response. A significant trend (P < 0.05) was only reported where none of the pairwise comparisons was significant.
The number of implantation sites, number of pups born, percentage of male pups Day 1 and pup weights were analysed using non-parametric methods. The non-parametric methods employed were the Kruskal-Wallis ANOVA, the Terpstra-Jonckheere test for a dose related trend and the Wilcoxon rank sum test for pairwise comparisons. Where the Kruskal-Wallis ANOVA was not significant, the pairwise comparisons were not reported in order to protect the Type I error.
The proportions of females with post-implantation survival index, live birth index and viability indices of 100 % and gestation, mating, fertility and fecundity indices were analysed using the Cochran-Armitage test for dose-response and Fisher’s exact test for pairwise comparisons. The tests were interpreted with one-sided risk for decreased incidence with increasing dose.

Reproductive indices:

Calculations
Group mean values for certain litter parameters were calculated as:
Mean 1 includes data from all surviving females giving birth including those showing total litter loss post-partum.
Mean 2 includes data from females rearing some young to weaning.

A number of indices were used, where appropriate, to evaluate reproductive function:

Mating index = (number of females with determined copulations / number of oestrous cycles required for their insemination) x 100

Female fecundity index = (number of pregnant females / number of females mated) x 100

Male fecundity index = (number of males siring one or more pregnancies / number of males with one or more confirmed matings) x 100

Female fertility index = (number of pregnant females / number of females paired) x 100

Male fertility index = (number of males siring one or more pregnancies / number of males paired) x 100

Median pre-coital time = time (day) by which half the females in the group had shown evidence of mating

Gestation index = (number of females with live pups / number of pregnant females) x 100

Post implantation survival index = (number of pups born / number of implantation sites) x 100

Live birth index = (number of pups alive Day 1 / number of pups born) x 100

% male pups (sex ratio) = (number of male pups Day 1 / number of pups of determined sex) x 100

Offspring viability indices:

Viability index 1 = (number of pups alive Day 4 before culling / number of pups alive Day 1) x 100

Viability index 2 = (number of pups alive Day 7 / number of pups alive Day 4 after culling) x 100

Viability index 3 = (number of pups alive Day 14 / number of pups alive 7) x 100

Viability index 4 = (number of pups alive Day 21 / number of pups alive 14) x 100

Clinical signs:

no effects observed

Description (incidence and severity):

The nature and intergroup distribution of the clinical observations showed no treatment-related trends.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no deaths in the P generation.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight gain of the high dose males was lower than that of the controls during the first five weeks of treatment and the difference was statistically significant during the pre-pairing period (P < 0.01, Dunnett’s test). Thereafter, growth of the males was essentially similar in all groups.
There was a dose-related reduction in the mean body weight gain of the females during the pre-pairing period and the difference from control was statistically significant in the high dose group (P < 0.01 Dunnett’s test). This overall lower mean body weight gain of the treated females during the pre-pairing period was reflected in lower initial mean body weights on Day 0 of gestation. During gestation, there was a dose-related reduction in mean body weight gain and in the high dose group, the difference from control was statistically significant between Days 0 and 7 (P < 0.001), Days 7 and 14 (P < 0.01) and Days 14 and 20 (P < 0.05, Dunnett’s test).
Again, the lower overall mean body weight gain of the treated groups during gestation resulted in lower mean body weights on Day 1 of lactation, particularly in the high dose group. However, during lactation, body weight gain was generally similar in all groups until the last week when the females in the treated groups gained less weight than the controls and the difference was statistically significant in the high dose group (P < 0.01, Dunnett’s test).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In the intermediate and high dose groups, there was a dose-related reduction in male mean food intake of the males throughout the pre-pairing period.
Generally, females in all treated groups ate less than the controls during the pre-pairing period and group mean food intake of the high dose females was markedly lower than control. During gestation and lactation, females in the high dose group ate significantly less than the controls (P < 0.01, gestation, P < 0.05, lactation, Dunnett’s test).

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Mating performance was unaffected by treatment.
The mean duration of gestation was unaffected by treatment. There was a statistically significant, dose-related, reduction in the mean numbers of implantation sites in all treated groups compared to control (P < 0.01, low and intermediate groups; P < 0.001, high dose group, Wilcoxon rank sum test). However, the values for the control, low and intermediate dose groups were higher than expected from the historical control data. Although the mean numbers of pups born were lower in all treated groups compared to control, again, the numbers in the control, low and intermediate dose groups were higher than expected from the background data. Given the small group sizes, the biological significance of the reductions in implantations and pups born in the low and intermediate dose groups is questionable. In the high dose group, the mean number of pups born was significantly lower than control (P < 0.01, Wilcoxon rank sum test). There was no effect of treatment on post-implantation survival index.
One female in each of the low and intermediate dose groups showed total litter deaths but the viability of the offspring in the treated groups was generally similar to that of the controls.
The mean body weight of the treated pups was similar to the controls on Day 1 post partum. Mean body weight gain of the pups in the treatment groups was slightly lower than that of the controls, particularly in the high dose group between Days 7 and 21 post partum, but there was no dose-relationship or statistical significance.
Necropsy examination of the weanling offspring showed only one male pup from the high dose group with slight, unilateral, renal pelvic dilatation.

Dose descriptor:

NOAEL

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

not determinable

Critical effects observed:

no

Clinical signs:

not specified

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not specified

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Although the mean body weights of the F1 males in the high dose group was only slightly lower than that of the controls at the start of this generation, mean body weight gain was significantly lower over the 4 weeks (P < 0.01, Dunnett’s test). Mean body weight gain of the F1 females in the high dose group was only marginally lower than that of the controls and the difference was not statistically significant.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Group mean food intake in the high dose group for both F1 males and females was slightly lower than the controls during the 4 weeks of this generation, particularly the females during the last week.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not specified

Anogenital distance (AGD):

not specified

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

F1

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

not determinable

Critical effects observed:

no

Reproductive effects observed:

yes

Lowest effective dose / conc.:

59.6 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

not specified

Relevant for humans:

not specified

Compound Consumption

The following mean intakes (mg/kg/day) of test material were achieved in the P generation.

Dose Level (ppm)	500	800	1200
Males pre-pairing	34.5	53.7	82.9
Females pre-pairing	41.0	64.7	98.4
Females gestation	38.2	60.6	88.8
Dose level (ppm)	300	530	790
Females lactation	48.1	85.8	130.2
Mean female	42.4	70.4	105.8
Sexes combined	38.5	62.1	94.4

 

The following mean intakes (mg/kg/day) of test material were achieved in the F1generation:

Dose Level (ppm)	500	800	1200
Males	59.6	98.0	148.4
Females	61.1	101.5	147.7
Sexes combined	60.4	99.8	148.1

 

Conclusions:

Under the conditions of the study dietary administration of 1 200 ppm of the test material to adult male and female rats for 10 weeks prior to mating, 790 ppm to the female rats during the lactation period, and again giving 1 200 ppm to the F1 generation for 4 weeks, produced slight adult and pup toxicity in terms of lower body weight gains and food intakes. At these concentrations there were fewer implantation sites compared to control.
At 500 and 800 ppm (reduced to 300 and 530 ppm during lactation), there were marginal effects on adult and pup weight gain and food intake and slightly lower numbers of implantation sites compared to control.
It is considered that reduction of dietary concentration by approximately one third during the lactation period in a 2-generation study, would allow demonstration of a maternal MTD without inducing excessive effects in the offspring.

Executive summary:

The reproductive toxicity of the test material was assessed according to OECD test Guideline 415 and in compliance with GLP.

The objective of the study was to provide a preliminary evaluation of the test material on the reproduction toxicity in the rat, and to define a Maximum Tolerated Dose (MTD) for a possible subsequent 2-generation study. As the compound was known to show a relatively steep dose relationship, dietary inclusion rates were amended during lactation to take account of the increased maternal food consumption at that time.

Groups of 12 male and 12 female rats were given the test material by dietary administration at nominal concentrations of 0 (control), 500, 800 and 1 200 ppm. Both sexes were treated for 10 weeks before pairing on a one to one basis within each group. The females continued with this treatment throughout gestation and then were allowed to litter and rear their offspring to weaning. During the lactation period, the females were given diets containing nominal concentrations of 0 (control), 300, 530 and 790 ppm the test material. Males were treated until evaluation of the females had been completed. Ten animals of each sex were randomly selected from each group to form the filial (F1) generation. These animals received the test material at the original concentrations for 4 weeks post-weaning.

Analysis of samples from the formulations prepared in Weeks 1, 11, 17 and 22 of the study showed that they were suitable for administration to the study animals.

P generation:

All animals survived to the scheduled kill.

There were no clinical observations that were related to treatment. Males in the high dose group gained less weight than the controls during the first 5 weeks of treatment. There was a dose-related reduction in female body weight gain during the pre-pairing period and gestation. Females in the high dose group gained less weight than the controls during the last week of the lactation period. Group mean food intake was lower than control in the intermediate and high dose group males and in the high dose females during the pre-pairing period. During gestation and lactation, females in the high dose group ate significantly less than the controls.

Mating performance and fertility were unaffected by treatment.

Mean duration of gestation was similar in all groups. In the high dose group, there was a reduction in mean number of implantation sites.

Pup viability was unaffected by treatment. Mean pup weight gain was slightly lower in the high dose group compared to control.

F1 generation:

Group mean body weight gain and food intake of the males and females in the high dose group were lower than control over the 4 week treatment period.

 

Under the conditions of the study dietary administration of 1 200 ppm of the test material to adult male and female rats for 10 weeks prior to mating, 790 ppm to the female rats during the lactation period, and again giving 1 200 ppm to the F1 generation for 4 weeks, produced slight adult and pup toxicity in terms of lower body weight gains and food intakes. At these concentrations there were fewer implantation sites compared to control.

At 500 and 800 ppm (reduced to 300 and 530 ppm during lactation), there were marginal effects on adult and pup weight gain and food intake and slightly lower numbers of implantation sites compared to control.

It is considered that reduction of dietary concentration by approximately one third during the lactation period in a 2-generation study, would allow demonstration of a maternal MTD without inducing excessive effects in the offspring.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because a pre-natal developmental toxicity study is available

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
In accordance with column 2 of REACH Annex VIII, a screening study for reproductive/ developmental toxicity, one species, does not need to be conducted if a pre-natal developmental toxicity study is available. Since two pre-natal developmental toxicity studies are available for the substance (rat and rabbit), it is considered justified to waive the requirement for a screening study for reproductive/ developmental toxicity.

Reference 3

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 June 1989 to 17 April 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Directive 87/302/EEC of November 18, 1987 adapting to technical progress for the ninth time Council Directive 67/548/EEC, pp. 47 - 50.

Version / remarks:

1988

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

Version / remarks:

1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japan/MAFF, Testing Guidelines for Toxicology Studies pp. 45 - 48.

Version / remarks:

1985

Deviations:

no

GLP compliance:

yes

Limit test:

no

Justification for study design:

- Route of administration: Since the test material is to be used as a herbicide and uptake of crop bearing residues by man cannot be ruled out, oral administration (via the diet) was selected.

Species:

rat

Strain:

Wistar

Remarks:

Chbb = THOM (SPF)

Details on species / strain selection:

This strain was selected since extensive experience is available on Wistar rats and the rat is the preferred animal species for reproduction studies according to the different test guidelines.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: Not specified
- Age at study initiation: F0 generation parental animals: 27 (± 1) days old when they arrived from the breeding facilities; 35 (± 1) days old at the beginning of treatment.
- Weight at study initiation: F0 generation parental male animals 126.2 g (117 - 134g), female animals 114.7 g (106 - 122 g).
During the acclimatisation period, animals with an extreme body weight were eliminated and sacrificed.
- Housing: During the study period, the rats were housed individually in type DKIII stainless steel wire mesh cages supplied by Becker & Co., Castrop-Rauxel., FRG (floor area of about 800 cm^ 2 ), with the following exceptions: During mating periods, the males designated for mating were kept individually in Makrolon cages, type M Ill (floor area of about 800 cm ^2 ); for the overnight mating the females were put into the cages of the males. From day 18 of pregnancy until day 14 after birth, the pregnant animals and their litters were also housed in Makrolon type M III cages. The M III cages were also suppl.ied by BECKER & CO. Pregnant females were provided with nesting material (cellulose wadding) toward the end of pregnancy.
- Diet: Ad libitum, except those animals selected for urinalyses had no access to food in the cages used to take urine samples.
- Water: Ad libitum, except those animals selected for urinalyses had no access to water in the cages used to take urine samples.
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Central air conditioning guaranteed a range of temperature of 20 - 24 °C. There were no deviations from these limits.
- Humidity (%): Central air conditioning guaranteed a range of relative humidity of 30 - 70 %. There were no deviations from these limits.
- Photoperiod (hrs dark / hrs light): The day/night rhythm was 12 hours (12 hours light from 6.00 to 18.00 hours and 12 hours darkness from 18.00 to 6.00 hours) in general.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: The test material preparations were prepared at intervals of not more than 35 days.
- Mixing appropriate amounts with: The test material preparations were prepared by weighing in the test material and mixing this thoroughly with a small amount of the food. This mixture was then mixed in a BOSCH household mixer. An appropriate amount of food was then added to obtain the desired concentration, and mixing was carried out for about 10 minutes in a GEBR. LÖDIGE laboratory mixer.

Details on mating procedure:

Matings of F0 and F1 generation parental animals
- M/F ratio per cage: At least 70 days after the beginning of treatment, males and females were mated at a ratio of 1:1 (generally male No. 1 with female No. 101, male No. 2 with female No. 102 etc.).
Throughout the mating period, each female animal was mated with a male animal that had been determined before.
- Length of cohabitation: Overnight. Normally, the female animal was placed toward 16.00 hours in the cage with its male partner and was again separated from the male animal between 7.00 and 9.00 hours of the following morning. Deviations from the specified times were possible on weekends and public holidays.
- Proof of pregnancy: A vaginal smear of the female animals was prepared after each mating and examined for sperm. If sperms were detected, the partners were no longer mated with each other. The day on which sperm were detected was denoted "day 0" and the following day "day 1" p.c. (post coitum).
- Further matings after two unsuccessful attempts: Yes. Animals was mated overnight for a maximum of 3 weeks.

Reevaluation of fertility
If an animal had not produced any offspring after the 2 matings (F1a and F1b litters) or after the mating for the F2 generation, those animals treated with the test material were mated with fertile animals of the control groups. Animals of the control groups which seemed to be infertile were mated with a mating partner with proven fertility of the controls.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical investigations to determine the active ingredient content, the stability and homogeneity of the test material were carried out before the beginning of the study, during the study period and after the end of the study. Furthermore, the test material was investigated for any additional constituents and trace contaminants.
For the determinations of the homogeneity representative samples were taken from the top, the middle and the bottom of the containers.
Analyses of the stability of the test material in the diet over 33 days was carried out before the start of the study for another rat feeding study.
The homogeneity of the test material in the maintenance diet was analytically investigated before the start of the study in the aforementioned rat feeding study and in addition for this reproduction study at the beginning of the study period.
In order to check the correctness of the concentration, samples of each one of the doses were sent for analysis to the analytical laboratory at the beginning of the study and then at approximately three-monthly intervals.
The content of the test material in the test material/food mixes was determined by HPLC-method.

The stability of the test material in the maintenance diet over a 33-day period was confirmed. The homogeneous distribution of the test material in the rat food used was also confirmed. According to the results of the concentration control analyses the minimum content of the test material in the food was generally guaranteed.

Duration of treatment / exposure:

The duration varied between generations. The study was conducted for approximately 44 weeks.

Frequency of treatment:

The F0 generation parental animals continuously received the test material at the appropriate dose levels via the diet until or up to about 16 hours before they were sacrificed, with the exception of infertile animals and those animals which were taken for urinalyses.

Details on study schedule:

F0 generation:
The females were allowed to litter and rear their pups (F1a generation pups) until day 4 or 21 after parturition. At least 10 days after the last weaning of the F1a generation pups, the F0 generation parental animals were mated again at a ratio of 1:1. The male partners were selected by lot, thus ruling out the possibility that mating was carried out with the same partners which produced the F1a generation pups.
The females were allowed to litter and rear their pups (F1b generation pups) until day 4 or 21 after parturition.
After the F1b generation pups had been weaned, the fasted (about 16 hours) F0 generation parental animals were sacrificed (with the exception of animals which were taken for the reevaluation of their fertility).

F1 generation parental animals and their progeny:
After weaning, 25 males and 25 females (each litter was taken into account) of the F1a pups were chosen per group (by lot) as the basis of the F1 generation parental animals. If fewer than 25 litters were available for selection or if one sex was missing in a litter, more animals were taken from different litters from the relevant test group to give the full number.
These animals were exposed continuously to the test material from their growth into adulthood until or up to about 16 hours before they were sacrificed (with the exception of infertile animals and those animals which were taken for urinalyses). At least 98 days after formation of the F1 generation parental animals, the males and females were mated at a ratio of 1:1. The male partners were selected by lot avoiding any mating between siblings.
The females were allowed to litter and rear their pups (F2 generation pups) until day or 21 after parturition.
After the F2 generation pups had been weaned, the fasted (about 16 hours) F1 generation parental animals were sacrificed (with the exception of animals which were taken for the reevaluation of their fertility).

Infertile animals:
If an animal of the F0 (after both mating intervals) or F1 generation parental animals had not produced any offspring, these animals were again mated for not more than 3 weeks with one fertile male or female animal of the control group each.
Thereafter the male animals were sacrificed shortly after sperm was detected in the vaginal smear of the relevant female partner. The relevant females were sacrificed before littering (if an animal after these matings had proved to be fertile) or about 10 days after the last mating (if an animal after these matings likewise had proved to be infertile).
Those parental animals from test groups 01, 02 and 03 (F0 generation parental animals) and 11, 12 and 13 (F1 generation parental animals) whose fertility had to be reevaluated, were offered test material-free diet only during the specific nightly matings (when they were kept together with one fertile animal of the control group). In the remaining times (i.e., the period between the individual matings as well as after the last mating until sacrifice), these animals were offered food with the test material depending to which group they belonged.

Dose / conc.:

20 ppm (nominal)

Dose / conc.:

100 ppm (nominal)

Dose / conc.:

500 ppm (nominal)

No. of animals per sex per dose:

25 animals per sex per dose for the F0 generation parental animals and F1 generation parental animals.

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The doses were chosen on the basis of various previous studies.
In a sub-chronic study, the test material was administered to 90 Wistar rats (45 males, 45 females) via their diet for 3 months. For comparison, a group of untreated animals (15 per sex) was used as control. The doses were 0, 50, 150, 450 ppm.
Food consumption and body weight were determined once a week; the rats’ state of health was checked each day. At the beginning of the study and toward the end of the administration period all the animals were subjected to ophthalmological examinations.
Two clinico-chemical and haematological examinations as well as two urinalyses were carried out. All animals were subjected to a gross-pathological examination. Finally, an extensive histopathological examination was carried out. The following findings were obtained and assessed or discussed in relation to the test material:
450 ppm group:
Clinico-chemical findings: Increase in the creatinine values in the plasma of female rats.
Organ weights: Increase of absolute and relative kidney weights in the animals of both sexes.
150 ppm group:
Organ weights: Increase of absolute and relative kidney weights in the male animals; increase of relative kidney weights in the female animals.
50 ppm group: No changes to which a test material-induced relevance can be attributed.

In a combined study three different doses (20, 100 and 400 ppm) of the test material were administered with the diet to 450 Wistar rats (225 males and 225 females) for 24 months. Each dose group was split up into a main group (50 animals per sex) and into the satellite groups I and II comprising 10 and 15 animals per sex, respectively. A group of untreated animals (75 males and 75 females) was maintained in parallel for comparison. It was likewise split into a main group and satellite groups I and II. The food consumption and body weight of the animals in the main group and of satellite group I was determined once a week up to 14 weeks and thereafter once a month until the end of the study. The state of health of all animals was checked daily; furthermore, the animals were subjected to additional inspection and palpation once a week. At the beginning of the study and then about every 6 months, animals of the main groups (control and highest dose) were examined ophthalmologically. Blood samples for haematological and clinico-chemical examinations were taken 5 times altogether from the animals of the satellite group II (at the last blood sampling, dead animals in this satellite group were supplemented by animals of the main groups). Urinalyses were carried out on the animals of satellite group I two times in the first year, and on 10 animals of each main group about 104 weeks after the beginning of the administration period. The animals that survived in satellite group I were subject to an interim kill after about 52 weeks; the rats surviving in the main group and satellite group II were sacrificed after about 104 weeks. All animals used were assessed by gross pathology. This was followed by a comprehensive histopathological examination. The following findings were obtained and assessed or discussed to be test material-related:
400 ppm: Significant increase in the relative kidney weights of the male rats of satellite group I and significant increase in the absolute and relative kidney weights of the males of the main group and satellite group II.
100 ppm: Significant increase in the relative kidney weights of the male rats of satellite group I and significant increase in the absolute kidney weights of the males of the main group and satellite group II.
20 ppm: No changes whatsoever that could be associated with the test material administered.

On the basis of the results of these studies, which indicated renal impairment being more pronounced in males than in females in dosages ranging from 450 ppm down to 100 ppm, the following doses were chosen for the reproduction study with the test material:
20 ppm: As the expected no adverse effect level.
100 ppm: As a concentration with possibly minimal toxic effects (e.g. increased kidney weights).
500 ppm: As the dose in which toxic effects were expected (e.g. kidney impairment) in the parental animals, but which should not induce mortality in these animals.

- Rationale for animal assignment: The assignment of the animals to the different test groups was carried out in accordance with a randomisation program, separately for males and females according to their weight, one day before the beginning of the administration period (day -1).
- Fasting period before blood sampling for clinical biochemistry: No

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily a check was made for any dead or moribund animals. If animals were in a moribund state, they were sacrificed and dissected like animals that died.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All parental animals were checked each day for clinically evident signs of toxicity. For technical reasons, however, the clinical observations recorded during the premating periods were printed out on a weekly basis
The nesting, littering, and lactation behaviour of the dams were generally evaluated in the mornings in connection with the daily clinical inspection of the dams. Only if there were any special findings (e.g., animal could not litter, umbilical cord not cut), these specific findings were documented with the dam concerned.
The littering behaviour of the dams was also inspected on weekdays (except holidays) in the afternoons in addition to the evaluations in the mornings. The day of littering was considered the period between about 15.00 hours of the one, and about 15.00 hours of the following day (= 24 hours). Deviations from this procedure were possible on Saturdays, Sundays and on public holidays, when the weighings of the dams took place as early as about 7.00 hours.
If an animal was not able to litter or was in a moribund state, these animals were sacrificed and examined in the laboratory of pathology.

BODY WEIGHT: Yes
- Time schedule for examinations: In general, the body weight of the male and female parental animals was determined once a week at the same time of the day in each case (in the morning); if possible, the weighings were carried out until the end of the study. The body weight change of the animals was calculated from these results. As exceptions, the following particularities applied to the female parental animals:
a) During each mating period (1st and 2nd matings of the F0 and mating of the F1 generation parental animals) the females were weighed on the day of positive evidence of sperm (day 0 p.c.) and on days 7, 14 and 20 post coitum.
b) Females showing no positive evidence of sperm within one mating period were not weighed during this mating interval.
c) Females with litters were weighed on the day of parturition (day 0 p.p.) and on days 4, 7, 14 and 21 postpartum.
d) Females without litters were not weighed during the lactation period of the dams used in parallel.
e) After weaning of the last F1a pups the female F0 generation parental animals were weighed weekly until the beginning of the 2nd mating interval (in parallel to the male F0 generation parental animals).
Furthermore, male and female animals whose fertility had to be reevaluated and those controls which were chosen as partners for these reevaluations were not weighed during the additional matings and the interval until they were sacrificed

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes.
- During the first 10 (F0 generation parental animals) and 14 (F1 generation parental animals) test weeks food consumption was determined once a week (each time for a period of 7 days) for males and females. After the 10th (F0 generation parental animals) and 14th (F1 generation parental animals) test week food consumption of the females during pregnancy (animals with evidence of sperm) was determined for days 0 - 7, 7 - 14 and 14 - 20 p.c..
- During the lactation period (animals with litter) food consumption was determined for days 0 - 4, 4 - 7 and 7 - 14 p.p..
- Food consumption was not determined between days 14 and 21 after parturition, since during this time pups will begin to consume considerable amounts of the rat food offered, and therefore there was no point in such a measurement.
- Food consumption of the males was not determined any longer after the 10th (F0 generation parental animals) or 14th (F1 generation parental animals) test week until sacrifice. Furthermore, there was no determination of food consumption in the females during the mating period, animals without positive evidence of sperm (during the gestation period of the dams used in parallel) and animals without litter (during the lactation period of the dams used in parallel).
- The food consumption of those animals whose fertility had to be reevaluated and those controls which were chosen as partners for these reevaluations was not determined either during the additional matings or the interval until they were sacrificed
- The intake of test material was calculated from the amount of food consumed and expressed in mg/kg body weight per day.
- The calculation of the group values/day was carried out according to the following formula:

(FC x D) / BWx = intake of test substance in mg/kg body weight/day

Where:
FC: Mean daily food consumption in grams (on day x + y; y = 3, 4, 6 or 7 depending on the study period).
D: Dose in ppm.
BWx: Mean body weight on a specific day in g x (on day x).

CLINICAL CHEMISTRY AND URINALYSES
Blood was taken from the retroorbital venous plexus in the morning from non-fasted, not anesthetised animals. The blood sampling procedures and the subsequent analysis of the serum samples were carried out in a randomised sequence. The list of randomisation instructions was compiled with a computer using a random number generator. For urinalyses the individual animals were transferred to metabolism cages and urine was collected at about 4 °C overnight. The urine samples were evaluated in a randomised sequence.
Male and female F0 parent urine collection was on nominal days 190, 191 and 197.
Male and female F0 parent blood collection was on nominal days 204/205.
Male and female F1 parent urine collection was on nominal days 152, 153 and 154.
Male and female F1 parent blood collection was on nominal days 155 and 158.
The assays of serum parameters were performed under internal laboratory quality control conditions with commercial reference controls to assure reliable test results.
The results of the clinico-chemical examinations are expressed in units of the International System (SI). The following examinations were carried out in 12 animals per test group and sex of the F0 and the Fl parental generation.

- Clinico-chemical examinations: An automatic analyser (Hitachi 737; by Boehringer, Mannheim, FRG) was used to examine the clinico-chemical parameters.
The following parameters were determined:
Enzymes: Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase.
Blood chemistry: Sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium.

- Urinalyses: With the exception of the sediment examination and the specific gravity, all the urine constituents were determined semi-quantitatively using test strips (Combur-9-test RL, by Boehringer, Mannheim, FRG) and a reflection photometer (Urotron RL9 model by Boehringer, Mannheim, FRG).
The specific gravity was determined using a urine refractometer.
The sediment was evaluated microscopically.
The following examinations were carried out: Volume, appearance, nitrite, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes, standardisation of litters F1a, F1b and F2 generation pups.
- The individual litters were standardised in such a way that, where possible, each litter contained 4 male and 4 female pups (always the first 4 pups/sex and litter were taken for further rearing). If it was not possible in single litters to have 4 pups/sex, it was proceeded in such a way that 8 pups per litter were present for further rearing (e.g., 5 male and 3 female pups). Standardisation of litters was not performed in litters with < 8 pups.
- Excess pups: With the exception of the F1a generation pups, which were chosen as the basis of the F1 generation parental animals, all pups were sacrificed (by means of CO2) after standardisation or weaning.

PARAMETERS EXAMINED
- Pup number and status at delivery: All pups derived from the F0 parents (F1a and F1b litters) and the F1 parents (F2 litter) were examined as soon as possible on the day of birth to determine the total number of pups and the number of liveborn and stillborn members of each litter. Pups which died before the first determination of their status on the day of birth were designated as stillborn pups.
- Pup viability/mortality: A daily check was made for any dead or moribund pups. The number and percentage of dead pups on the day of birth (day 0) and of pups dying between days 1 - 4, 5 - 7, 8 - 14 and 15 - 21 of the lactation period were determined. However, pups which were culled or died accidentally were not included in these calculations. The number of live pups/litter was calculated on the day of birth, and on lactation days 4 (before and after standardisation of litters), 7, 14 and 21.
- Sex ratio: On the day of birth (day 0) and on the first few days after birth the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle: Normally, the anogenital distance is considerably greater in male than in female pups.
During the remaining rearing period the sex of the pups was assessed by external appearance and the sex was finally confirmed at necropsy.
- Pup body weight data: The pups were weighed on the day after birth (day 1 p.p.) and on days 4 (before standardisation), 7, 14 and 21 after birth. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning) and on day 4 p.p. before standardisation of the litters.
- Pup clinical observations: The pups were examined each day for clinical symptoms (including gross-morphological findings).
- Development stages: Different development stages of the pups were monitored. A physiological development was assumed if the following parameters had been fulfilled on the days given in brackets:
Pinna unfolding (on day 4 after birth before standardisation)
Opening of the auditory canal (on day 13 after birth)
Opening of the eyes (on day 15 after birth)
The examinations to establish whether the various development stages had been reached were carried out in the morning in each case.

GROSS EXAMINATION OF DEAD PUPS:
With the exception of the F1a generation pups, which were chosen as the basis of the F1 generation parental animals, all pups were sacrificed (by means of CO2) after standardisation or weaning. These pups, including stillborn pups and those that died during their rearing period, were subjected to a macroscopic (external and organ) examination. Thereafter, stillborn pups and those that died during the rearing period as well as pups which were culled on day 4 p.p. or "surplus" (i.e. those pups with scheduled sacrifice after weaning) pups which showed any remarkable findings during rearing or abnormalities in the macroscopic assessment were treated with different methods.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:
Behavioral tests
Up to weaning, different behavioral tests with the pups were performed in order to record any variations from normal. All surviving pups were tested for the gripping reflex, the hearing test (acoustic startle) and the pupillary reflex test.
- Gripping reflex: On day 13 after birth (± 1 day), the pups were checked for this reflex. For this purpose, the front extremities of the animals were placed on a bar about 3 mm in diameter. The animals had to cling to this bar and pull themselves up. If this aim was achieved, the test was regarded as positive (yes response), if it was not, the test was regarded as negative (no response).
- Hearing test (acoustic startle): On day 21 after birth (± 1 day), the animals were placed in a soundproof box (internal dimensions 49.5 x 49.5 x 38.5 cm). After a short acclimatisation period, the animals were exposed (twice at most) to a sound (0.1 seconds, 5 000 Hz, about 90 dB). A startle reflex (movement of the ears or a jerk) was considered to be a response to this stimulus.
- Pupillary reflex (pupil constriction): On day 20 after birth (± 1 day), the animals pupils were allowed to dilate by introducing the pups to a low light environment. The pupillary constriction reflex was assessed by shining a pencil torch on the eye and observing the reaction of the pupil. Miosis was assessed as the presence of the reflex.

Postmortem examinations (parental animals):

SACRIFICE
The animals were sacrificed by decapitation under CO2 anesthesia. Then the animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs. Animals which died intercurrently or were sacrificed in a moribund state were necropsied as soon as possible after their death and assessed by gross pathology.

GROSS NECROPSY
Reevaluation of fertility: After fertility had been reevaluated, the animals were sacrificed and subjected to gross-pathological assessments.
- Reevaluation of fertility: The uteri (including the contents) of the relevant female animals were assessed by the reproduction toxicology laboratory. If macroscopic implantations were seen, their number, position and condition were recorded. Uterus contents were then removed as completely as possible. The uteri of apparently non-pregnant animals, or empty uterus horns were placed in 10 % ammonium sulfide solutions for about 5 minutes in order to be able to recognise early resorptions or implantations. Then the uteri were rinsed carefully under running water. After these examinations of the uteri were completed at the reproduction toxicology laboratory, the uteri of the corresponding animals were transferred to the pathology lab for further fixation and evaluation.

The weight of the anesthetised animals and the weight of the liver, kidneys, and testes from all animals sacrificed at scheduled dates were determined.
Subsequently, the following organs or tissues of the F0 and F1 generation parental animals were fixed in 4 % formaldehyde solution:

Control and 500 ppm groups: Vagina, cervix, uterus, ovaries, testes, epididymides, seminal vesicles, coagulating gland, prostate gland, pituitary gland, liver, kidneys, all gross lesions.
For the 20 ppm and 100 ppm groups only the liver, kidney and gross lesions were examined.
Of the animals that were necropsied prematurely, the above mentioned organs were processed and investigated histopathologically.

Postmortem examinations (offspring):

SACRIFICE
Culled (i.e. all pups which were sacrificed on day 4 p.p. as a result of standardisation) and surplus pups (i.e. all pups reared until day 21 p.p. except those F1a generation pups selected as the basis of the F1 parental generation) were sacrificed on day 21 after birth or subsequent days by means of CO2.

GROSS NECROPSY
- Pups were examined externally and eviscerated, and their organs were assessed macroscopically. If there were notable findings or if abnormalities were found in the daily clinical observation of the animals after their delivery, the affected animals were, if it was deemed necessary, examined additionally using appropriate methods (e.g., skeletal staining according to modified Dawson’s method and/or further processing of head according to Wilson's method).
- All stillborn pups and all pups that died during rearing were examined externally and eviscerated, and their organs were assessed macroscopically. If there were notable findings or if abnormalities were found in the daily clinical observation of the animals after their delivery, the affected animals were, if it was deemed necessary, examined additionally using appropriate methods (e.g., skeletal staining according to modified Dawson's method and/or further processing of head according to Wilson's method).
- The stained skeletons were evaluated under a stereomicroscope or a magnifying glass.
- All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation.

Statistics:

- Clinical examinations: Dunnett’s Test was used for statistical evaluation of all parametric data such as food consumption (parental animals), body weights and body weight change (parental animals and pups). Fisher's Exact Test was used for statistical evaluation of all quantitative data such as developmental stages, gripping and pupillary reflex, hearing test, number of live and dead pups, and the different indices (e.g. male and female mating index, male and female fertility index, gestation index, live birth index, viability index, lactation index). Differences between control and treated groups were considered significant at p ≤ 0.05 or p ≤ 0.01.
- Clinical chemistry: Means and standard deviations have been calculated for each test group and tabulated together with the individual values. In order to test if the results of the individual dose groups differed statistically significantly from the results of the control group, the means for the dose groups were compared with those for the control group using the analysis of variance (ANOVA and Dunnett’s test).
- Urinalyses: The assessment to whether certain characteristics differed in degree in the control and test groups was carried out using the chi^2 test in appropriate two-by two contingency tables.

Reproductive indices:

Males
The mating partners, the number of mating days until vaginal sperm could be detected in the female mating partner, and the pregnancy status of the female partner were noted for F0 and F1 males. For the males, mating and fertility indices were calculated for F1a, F1b and F2 litters according to the following formulae:

Male mating index (%) = (number of males with confirmed mating† / number of males placed with females) x 100
† A female with vaginal sperm or that gave birth to a litter or with pups/foetuses in utero.

Male fertility index (%) = (number of males proving their fertility† / number of males placed with females) x 100
† A female giving birth to a litter or with pups/foetuses in utero

The mating partners, the number of mating days until vaginal sperm could be detected, and pregnancy status were recorded for F0 and F1 females. For the females, mating, fertility and gestation indices were calculated for F1a, F1b and F2 litters according to the following formulae:

Females:
Female mating index (%) = (number of females mated† / number of females placed with males) x 100
† The number of females with vaginal sperm or that gave birth to a litter or with pups/foetuses in utero.

Female fertility index (%) = (number of females pregnant† / number of females mated‡) x 100
† The number of females that gave birth to a litter or with pups/foetuses in utero.
‡ The number of females with vaginal sperm or that gave birth to a litter or with pups/foetuses in utero.

Gestation index (%) = (number of females with live pups on the day of birth / number of females pregnant†) x 100
† The number of females that gave birth to a litter or with pups/foetuses in utero.
Furthermore, the total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1a, F1b and F2 litters according to the following formula:

Live birth index (%) = (number of liveborn pups at birth / total number of pups born) x 100

Offspring viability indices:

Viability and lactation indices were calculated according to the following formulae:

Viability index (%) = (number of live pups on day 4† after birth / number of liveborn pups on the day of birth) x 100
† Before standardisation of litters (i.e. before culling).

Lactation index (%) = (number of live pups on day 21 after birth / number of live pups on day 4† after birth) x 100
† After standardisation of litters (i.e. after culling).

The sex ratio was calculated for day 0 and day 21 after birth according to the following formula:

Sex ratio = (number of live male or female pups on day 0/21 / number of live male and female pups on day 0/21) x 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical observations for males and females (except gestation and lactation periods):
No clinical signs which might be attributed to the test material administered were detected in male or female F0 generation parental animals. The 3 doses (20, 100 and 500 ppm) administered as addition to the diet did not lead to disturbances of the general behavior in any of the F0 parental animals.
Only some spontaneous findings without any relation to dose occurred in a few animals of all groups including the controls. Mainly minor skin lesions, swelling of limbs or alopecia were found; furthermore, one control male showed a palpable mass at the flank towards the end of the study and for one 100 ppm male broken incisivi and chromodacryorrhea, which was also noted for one high dose female, were recorded.

Clinical observations for females during gestation:
There were no particular test material-related clinical findings in F0 females during gestation periods for F1a or F1b litters. Without any dose-response relationship insufficient/no nesting activity was observed for several dams of all groups during gestation (F1a and F1b). Moreover, during the gestation period for the F1b pups blood in bedding was found in one high dose female and haemophthalmia in one 100 ppm dam, which died subsequently.

Clinical observations for females during lactation:
There occurred no test material-related clinical findings in the dams during the lactation of F1a and F1b pups. Only some spontaneous findings were recorded: Dam No. 150 (20 ppm group) showed a skin lesion of the left hindlimb during the lactation period of F1a litter.
One animal of the 100 ppm group (No. 167) died during delivery of F1b pups. Moreover, after parturition, umbilical cords were not cut in some pups and/or placenta(e) were not consumed by dams of different groups without showing any dose-response relationship (F1a and/or F1b).

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were 2 F0 females (Nos. 158 and 167 - 100 ppm) which died spontaneously; these deaths are not associated with treatment. Animal No. 158 died during gestation of F1b litter (day 10 p.c.) and dam No. 167 died during the delivery of F1b pups.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

During the whole study the mean body weights/body weight gains of the males and of the females (including gestation and lactation periods) do not show any influence of the test material on these parameters. All differences between the controls and the animals of the test material-treated groups are assessed to be within the expected range of biological variation. This includes the few isolated statistically significant differences concerning body weight gain between the male controls and the 500 ppm males (week 9 - 10) and the female control and the 100 ppm group (week 4 - 5, lactation days 14 - 21 (F1a) and week 18 - 19).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no differences of biological relevance between the controls and the test material-treated groups concerning the food consumption of the F0 males during the premating period and the food intake of the F0 females during premating, gestation and lactation periods.
The amount of test material (in mg) which was consumed by the animals per kilogram body weight each day was calculated at the times at which food consumption was determined.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test material induced changes were found in the clinico-chemical examinations in both sexes of F0 parental animals.
There are some statistically significant inter-group differences in the results of the clinico-chemical data. These deviations are marginal, inconsistent or incidental. Accordingly, these changes are considered to be of no toxicological significance.
Furthermore, the statistically significant increase in alkaline phosphatase activity seen in the high-dose males of the F0 parental animals is not considered to be test material-related, because the value of this treatment group (5.42 μkat/L) is close to the upper limit of the range of historical control data of male Wistar rats after a study period of 6 months (4.19 μkat/L - 5.05 μkat/L) and no corresponding findings were observed in the other animals of this dose group.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test material-induced changes were observed in the urinalyses in both sexes of F0 parental animals.
There are some statistically significant inter-group differences in the results of the urine data. These deviations are marginal, inconsistent or incidental. Accordingly, these changes are considered to be of no toxicological significance.

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

None of the histopathological findings were considered to be associated with the administration of the test material. There were two premature decedents in test group 02 (100 ppm): Female No. 158 which had a malignant schwannoma and female No. 167 for which no cause of death could be established on histological examination. There was no evidence to suggest that either of these deaths were related to administration of the test material. Neither is the bilateral degeneration of the germinal epithelium and the subsequent dystrophic mineralisation in the testes, and the aspermia observed in the epididymides of male animal No. 44 in the lowest dose group (20 ppm) considered treatment-related, but rather as an incidental finding.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Male Reproduction Data
For all F0 males (except No. 44, test group 01 - 20 ppm; mating for F1b pups) which were placed with females to generate F1a or F1b pups mating was confirmed so that the male mating index was 100 % in all groups (F1a and F1b) except for test group 01 (F1b) where it was 96 %.
Nearly all males (except 2 of the 20 ppm group) proved their fertility after the scheduled matings for F1a and/or F1b litters.
For 1 male of the 20 ppm group (No. 31) for which fertility could not be proven after mating for F1a or F1b litters, fertility was confirmed in the reevaluation of fertility; however, for another low dose male (No. 44) fertility could not be proven. The observable differences concerning the male fertility indices (for F1a and F1b) between the groups are finally assessed as being of spontaneous nature and not related to the test material administration. All values are in the range of the historical control.

Female Reproduction Data
The female mating indices calculated after the mating period for F1a and F1b litters was 100 % for all groups except for test group 01 (20 ppm), where it only reached 96 % for the second (F1b) mating period.
The mean duration until sperm could be detected (day 0 p.c.) varied between 2.4 and 2.7 days (F1a) or 2.1 and 2.8 days (F1b) without there being any differences of biological significance between the groups.
The female fertility indices, varied between 100 % and 88 % (concerning F1a) and between 100 % and 96 % (concerning F1b). However, for all dams except No. 166 (100 ppm) fertility was confirmed at least in one of the scheduled matings.
The mean duration of gestation (F1a and F1b) was similar in all groups.
Due to one dam of test group 02 (100 ppm) - No. 158 - which was pregnant, but died during the F1b gestation period, the gestation index calculated for test group 02 (100 ppm) was 96 %, while it was 100 % for all other groups (concerning F1a and F1b litters). The mean number of F1a and F1b pups delivered and the relevant live birth indices show no differences of biological relevance and no dose-related trend.

Re-evaluation of Fertility
2 males (Nos. 31 and 44 - 20 ppm) and 1 female (No. 166) of test group 02 (100 ppm) had to be re-evaluated. Fertility could be confirmed only for male No. 31.

Dose descriptor:

NOAEL

Effect level:

ca. 50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Dose descriptor:

NOAEL

Effect level:

ca. 10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

organ weights and organ / body weight ratios

Dose descriptor:

NOAEL

Effect level:

ca. 2 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

Critical effects observed:

yes

Lowest effective dose / conc.:

2 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

not specified

Dose response relationship:

yes

Relevant for humans:

not specified

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

F1 generation parental animals - Clinical observations for males and females
No clinical signs which might be attributed to the test material administered were obtained in male or female F1 generation parental animals of anyone test group during the relevant intervals (F1 males: Whole study period; F1 females: Premating and the interval after weaning of F2 pups until sacrifice: For clinical observations of the dams during gestation and lactation periods.
Only some spontaneous findings were recorded in single F1 males and females during premating like microphthalmia (male No. 217 - control group), chromodacryorrhea (male No. 272 - 100 ppm), malocclusion (males Nos. 247 - 20 ppm and 292 -500 ppm) and alopecia (female No. 378 - 500 ppm).

F1 generation parental animals - Clinical observations for females during gestation
No particular clinical findings were noted for F1 dams except no or insufficient nesting activity, which was recorded for several dams of all groups including the control and which occurred without any dose-response relationship.

F1 generation parental animals - Clinical observations for females during lactation
After parturition, only one dam (No. 358) of test group 12 (100 ppm) did not nurse its pups properly; all pups of this dam died until day 5 p.p..
No further clinical findings were seen.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

None of the F1 parental animals died intercurrently or had to be sacrificed prematurely.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights and body weight gains were not adversely affected by the administration of the test material to the parental animals of test groups 11, 12 and 13 (20, 100 and 500 ppm) during the whole study period including gestation and lactation periods of the dams for F2 litters. All differences between the controls and the test material-treated groups are regarded as spontaneous, including the sporadic and sometimes even statistically significantly increased or decreased body weight gains of the females in test groups 11 (20 ppm), 12 (100 ppm) and 13 (500 ppm) during premating, gestation and lactation, which occurred without any dose response relationship.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No impairment of food consumption was recorded for the test material-treated F1 parental animals when compared to the controls, neither during the premating period nor during the gestation and lactation period of the F1 females. All differences between the groups are without any biological relevance.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No test material induced changes were found in the clinico-chemical examinations in both sexes of F1 parental animals.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No test material-induced changes were observed in the urinalyses in both sexes of F1 parental animals.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Changes in the relative and absolute mean kidney weights were present in animals of both generations given 100 or 500 ppm. No morphological findings were present in the kidneys which could explain this organ weight change. Nonetheless the consistency of its occurrence suggests that it resulted from the administration of the test compound. Males of the F0 generation given the highest dosage had significantly lower mean relative liver weights than controls (p<0.05). However, this intergroup difference was attributed to chance since on light microscopy hepatic morphology was comparable and no corresponding change was seen in the F1 generation.

As with the F0 generation, mean kidney weights of animals given 100 or 500 ppm were significantly greater than control values. In males, statistically significant differences were attained in animals which had received 100 or 500 ppm for both absolute (p<0.05, p<0.01 respectively) and relative (p<0.01 for both groups) values while in females significant differences were noted at 100 and 500 ppm for relative weights (p<0.05, p<0.01 respectively) but only at 500 ppm for absolute weights (p<0.01).

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

F1 generation parental animals - Male reproduction data
For all F1 males of all test groups, which were placed with females to generate F2 pups, mating was confirmed (i.e. the male mating index for all groups was 100 %). Whereas all control males and the males of test group 11 (20 ppm) proved to be fertile, the fertility of two males of the 100 and of one male of the 500 ppm group could not be proven during the scheduled matings for F2 litters.
The fertility indices were:
100 % for test group 10 (all males proved their fertility)
100 % for test group 11 (all males proved their fertility)
92 % for test group 12 (2 out of 25 males without proving their fertility)
96% for test group 13 (1 out of 25 males without proving its fertility)
However, all males in question - except male No. 262 (100 ppm group) - proved their fertility later. Therefore, the differences concerning the fertility indices of F1 males are regarded as incidental.


F1 generation parental animals - Female reproduction and delivery data
The female mating index reached 100 % for all test groups. The mean duration until sperm could be detected varied between 2.2 and 3.2 days.
The female fertility index calculated was 100 % for test group 10 and 11, 92 % for test group 12 and 96 % for test group 13.
However, the differences concerning the female fertility index between the groups are assessed as being of spontaneous nature, because all relevant females proved heir fertility later on.
The mean duration of gestation (F2) was similar for all groups.
All pregnant females gave birth to litters with liveborn pups. Consequently the gestation index was 100 % for all groups.
The mean number of delivered F2 pups and the percentage of liveborn F2 pups show no substantial differences between the control and the test material-treated groups. Consequently, the live birth index is not substantially influenced.

F1 generation parental animals - Reevaluation of fertility
Several males and females of test groups 12 and 13 (100 and 500 ppm) had to be reevaluated concerning their fertility. All of them proved to be fertile when mated with a fertile female or male except one 100 ppm - male (No. 262) for which fertility could not be proven.
The mating partner of this male showed a positive evidence of sperm, but did not become pregnant.

Dose descriptor:

NOAEL

Effect level:

ca. 2 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Dose descriptor:

NOAEL

Effect level:

ca. 50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

F1a and F1b generation pups/litters
No abnormal clinical symptoms were recorded for the F1a and F1b pups. Only some clinical findings (e.g. shortened tail, lesion of hindlimbs and traumatic lesion of cornea) were detected in very few F1a and F1b pups of different groups without a clear dose response relationship. These findings are finally assessed as spontaneous ones.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

F1a and F1b generation pups/litters: Pup number and status at delivery
The mean number of delivered F1a and F1b pups/dam and the percentages of liveborn and stillborn F1a and F1b pups do not show any differences of biological relevance between the groups.

F1a and F1b generation pups/litters: Pup viability/mortality
The number of pups, which died or were cannibalised from the day of birth until day 4 p.p. (before culling) was slightly increased in the 500 ppm group (F1a and 1b) and also marginally increased in the 100 ppm group (F1b) in comparison to the control group.
Consequently, the viability indices as indicators of the pups' viability during the first 4 days after birth were lowest in this/these test group(s), but still in the range of the historical control. Finally, the impaired pup viability in test groups 03 (500 ppm - F1a and F1b) and 02 (100 ppm - F1b) is assessed as test material-related. Similar effects were also found for the high dose F2 pups. The lactation index as an indicator of offspring survival between days 4 - 21 p.p., however, was not influenced by the substance administration (F1a and F 1 b).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

F1a and F1b generation pups/litters
Only body weight gains of the high dose F1a pups were marginally impaired in comparison to the controls on days 7 - 14 p.p., which has to be attributed to the test material administration
The statistically significantly reduced weight gain of the 100 ppm F1b pups, however, is assessed as a spontaneous effect due to the missing dose-response relationship.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not specified

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

F1a and F1b generation pups/litters
The examination of stillborn pups, pups that died intercurrently, culled pups and surplus pups of F1a and F1b litters did not reveal any difference of biological relevance between the test groups either in the type or in the number of pup necropsy observations. The statistically significant increase of 20 ppm pups of the F1b generation, which showed findings at necropsy is mainly caused by a higher number of pups with dilated renal pelvis; however, this finding is also present in the historical control data at a comparable incidence. Only very few of the large number of examined pups of all groups showed some other spontaneous findings like incisors sloped, hydroureter and focal liver necrosis.

Results of the pathology examination indicated that the administration of the test material produced no morphological changes in animals of the F1 generation which could be attributed to the treatment with test material.
In comparison to controls, there was, in both generations, a statistically significant increase in the relative and absolute mean kidney weights of treated animals for which no correlating morphological finding was found at light microscopy.

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All the histomorphological findings were considered to have occurred fortuitously and were not associated with the administration of the test compound.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

F1a and F1b generation pups/litters - Sex ratio
The sex distribution and sex ratios of live F1a and F1b pups on the day of birth and on day 21 p.p. did not show any substantial difference between controls and treated test groups; all differences observed are regarded as spontaneous.

F1a and F1b generation pups/litters - Development stages
As to the several morphological development stages monitored up to weaning there were no biologically relevant differences between the control and the test material-treated F1a or F1b pups. The statistically significantly lower number of F1b pups of test groups 02 and 03 (100 and 500 ppm) with pinna unfolding on time and the statistically significantly higher number of low dose F1b pups with eye opening on time are not assessed as test material-related effects due to missing dose-response relationship.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

F1a and F1b generation pups/litters
Out of the different behavioral tests which the F1a and F1b pups had to undergo up to weaning, no remarkable differences, which could be assessed as test material-related, were noted.

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

F1a

Effect level:

ca. 10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

Dose descriptor:

NOAEL

Generation:

F1b

Effect level:

ca. 2 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

F2 generation pups/litters - Pup clinical observations
F2 generation pups did not show any clinical symptoms during rearing which could be attributed to the treatment. For a few pups some spontaneous findings like oedema of the hindlimbs, lesion of/or shortened tail and lesion of hind-and/or forelimbs were recorded without any dose-response relationship.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

F2 generation pups/litters - Pup number and status at delivery
The mean number of delivered F2 pups/dam and the percentages of liveborn and stillborn pups do not show any statistically significant differences between the groups. Although the number of stillborn pups seems to be increased in the test material-treated groups it is fully in the range of biological variation.

F2 generation pups/litters - Pup viability/mortality
The number of pups, which died or were cannibalised from days 0 - 4 p.p. (before culling) was increased in the 500 ppm group in comparison to the control group and as a consequence, the viability index as an indicator of the pups' viability during the first 4 days after birth was lowest in this test group; this has to be assessed as a test material-induced effect.
The statistically significantly increased number of cannibalised pups in the 20 ppm group, however, is regarded as an incidental finding, which was mainly caused by dam No. 337, which cannibalised already 7 out of the 16 pups cannibalised in total.
The lactation index as an indicator how pups were nursed during the rest of their rearing varied for F2 pups between 100 % (test group 11 - 20 ppm) and 97 % (test group 13 - 500 ppm) and did not show any substantial differences between the groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

F2 generation pups/litters - Pup body weight data
Mean pup body weights of the 500 ppm group are statistically significantly lower in comparison to the relevant control values on days 14 and 21 p.p.; moreover, weight gain of these pups is impaired on days 4 - 7, 7 - 14 and 4 - 21 p.p., which has to be attributed to the test material administration. All other differences between the groups concerning pup body weight data of the F2 generation are of spontaneous nature.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

F2 generation pups/litters - Pup necropsy observations
All recorded pup necropsy observations (e.g. incisors sloped, cataract, dilated renal pelvis etc.) occurred without a clear dose-response relationship. They were recorded for a very few pups of different groups with or without involvement of the control group and are assessed as being of spontaneous origin.

Histopathological findings:

not specified

Other effects:

effects observed, treatment-related

Description (incidence and severity):

F2 generation pups/litters - Sex ratio
The sex distribution and sex ratio of live F2 pups on the day of birth and on day 21 p.p. did not show any biologically relevant differences between controls and treated test groups. All differences observed are regarded as spontaneous.

F2 generation pups/litters - Development stages
As to the morphological development stages monitored up to weaning, the number of pups of test groups 11, 12 and 13 (20, 100 and 500 ppm) showing pinna unfolding on time was statistically significantly reduced in comparison to the control group; however, due to missing dose-response relationship and the unexpected high number of control pups with a positive test result, this is finally assessed as an incidental finding. This is also assumed for the lower number of pups of the intermediate dose group with a positive test result on eye opening.
The statistically significantly lower number of high dose pups, however, which showed auditory canal opening on time is probably connected with the retarded, test material-related growth of these pups.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

F2 generation pups/litters - Behavioral tests
No substantial differences could be noted between the F2 pups of test groups 11 - 13 (20, 100, 500 ppm) and the control pups the observable differences are without any biological relevance.

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

ca. 10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

other: Higher number of pups with delayed auditory canal opening.

Critical effects observed:

no

Reproductive effects observed:

no

Absolute Weights: Mean Values Males F0 Generation

Dose Group		0	1	2	3
Bodyweight	M (g)	552.083	541.461	543.52	539.312
	SD	40.489	38.125	62.586	39.376
	n	23	23	25	25
Liver	M (g)	18.058	17.514	17.06	16.484
	SD	2.244	1.946	3.138	1.884
	n	24	23	25	25
Kidneys	M (g)	3.376	3.38	3.492	3.694**
	SD	0.287	0.337	0.281	0.231
	n	24	23	25	25
Testes	M (g)	3.796	3.706	3.667	3.73
	SD	0.305	0.289	0.367	0.267
	n	24	23	25	25

* P ≤ 0.05; Dunnett test two-sided.

** P ≤ 0.01; Dunnett test two-sided.

 

Absolute Weights: Mean Values Females F0 Generation

Dose Group		0	1	2	3
Bodyweight	M (g)	314.226	314.368	315.305	313.304
	SD	14.516	29.748	19.369	21.353
	n	23	25	22	25
Liver	M (g)	9.783	9.849	9.49	9.888
	SD	0.754	1.371	0.819	0.97
	n	23	25	22	25
Kidneys	M (g)	2.098	2.12	2.157	2.286**
	SD	0.146	0.186	0.142	0.166
	n	23	25	22	25

* P ≤ 0.05; Dunnett test two-sided.

** P ≤ 0.01; Dunnett test two-sided.

 

Relative Weights: Mean Values Males F0 Generation

Dose Group		0	1	2	3
Bodyweight	M (%)	1000	100	100	100
	SD
	n	23	23	25	25
Liver	M (%)	3.244	3.232	3.124	3.053*
	SD	0.251	0.239	0.282	0.221
	n	23	23	25	25
Kidneys	M (%)	0.61	0.624	0.646*	0.687**
	SD	0.047	0.042	0.049	0.048
	n	23	23	25	25
Testes	M (%)	0.689	0.687	0.681	0.694
	SD	0.05	0.061	0.086	0.056
	n	23	23	25	25

* P ≤ 0.05; Dunnett test two-sided.

** P ≤ 0.01; Dunnett test two-sided.

 

Relative Weights: Mean Values Females F0 Generation

Dose Group		0	1	2	3
Bodyweight	M (%)	100	100	100	100
	SD
	n	23	25	22	25
Liver	M (%)	3.113	3.124	3.008	3.156
	SD	0.181	0.201	0.151	0.218
	n	23	25	22	25
Kidneys	M (%)	0.668	0.667	0.685	0.731**
	SD	0.043	0.054	0.039	0.051
	n	23	25	22	25

* P ≤ 0.05; Dunnett test two-sided.

** P ≤ 0.01; Dunnett test two-sided.

 

Absolute Weights: Mean Values Males F1 Generation

Dose Group		0	1	2	3
Bodyweight	M (g)	545.459	523.94	535.183	537.204
	SD	65.445	54.429	62.088	44.204
	n	22	25	23	24
Liver	M (g)	17.355	16.575	16.818	16.764
	SD	3.144	2.907	2.712	2.13
	n	22	25	23	24
Kidneys	M (g)	3.084	3.136	3.326*	3.562**
	SD	0.239	0.324	0.3	0.282
	n	22	25	23	24
Testes	M (g)	3.835	3.768	3.192	3.875
	SD	0.287	0.302	0.411	0.298
	n	22	25	23	24

* P ≤ 0.05; Dunnett test two-sided.

** P ≤ 0.01; Dunnett test two-sided.

 

Absolute Weights: Mean Values Females F1 Generation

Dose Group		0	1	2	3
Bodyweight	M (g)	295.318	297.38	282.787	293.513
	SD	19.516	25.787	13.526	16.87
	n	22	25	23	24
Liver	M (g)	9.41	9.534	9.02	9.712
	SD	0.765	1.048	0.456	0.776
	n	22	25	23	24
Kidneys	M (g)	2.03	2.107	2.048	2.189**
	SD	0.16	0.201	0.157	0.181
	n	22	25	23	24

* P ≤ 0.05; Dunnett test two-sided.

** P ≤ 0.01; Dunnett test two-sided.

 

Relative Weights: Mean Values Males F1 Generation

Dose Group		0	1	2	3
Bodyweight	M (%)	100	100	100	100
	SD
	n	22	25	23	24
Liver	M (%)	3.167	3.152	3.135	3.118
	SD	0.287	0.316	0.279	0.263
	n	22	25	23	24
Kidneys	M (%)	0.57	0.6	0.625**	0.664**
	SD	0.053	0.048	0.052	0.043
	n	22	25	23	24
Testes	M (%)	0.711	0.724	0.736	0.726
	SD	0.087	0.075	0.085	0.082
	n	22	25	23	24

* P ≤ 0.05; Dunnett test two-sided.

** P ≤ 0.01; Dunnett test two-sided.

 

Relative Weights: Mean Values Females F1 Generation

Dose Group		0	1	2	3
Bodyweight	M (%)	100	100	100	100
	SD
	n	22	25	23	24
Liver	M (%)	3.19	3.209	3.191	3.309
	SD	0.222	0.263	0.12	0.184
	n	22	25	23	24
Kidneys	M (%)	0.688	0.71	0.725**	0.746**
	SD	0.043	0.048	0.051	0.045
	n	22	25	23	24

* P ≤ 0.05; Dunnett test two-sided.

** P ≤ 0.01; Dunnett test two-sided.

Conclusions:

Under the conditions of the study, the NOAEL for reproductive effects was 500 ppm. The NOAEL for systemic toxicity of the test material was 100 ppm for F0 females, F1 and F2 pups, and 20 ppm for F0 males, for males and females of the P1 parental generation and the F1b pups.

Executive summary:

The reproductive toxicity of the test material was assessed according to OECD Test Guideline 416, JMAFF, EPA/FIFRA and European Commission guidelines and in compliance with GLP.

The test material was administered to groups of 25 male and 25 female immature rats (F0 parental generation) as a constant homogeneous addition to the food in different dose levels (0, 20, 100 or 500 ppm). At least 70 days after the beginning of treatment, F0 animals were mated to produce a first litter (F1a) and subsequently re-mated to produce a second litter (F1b retained only until weaning). Groups of 25 males and 25 females selected from F1a pups as F1 parental generation were offered diets containing 0, 20, 100 and 500 ppm of the test material post weaning, and the breeding program was repeated to produce F2 litter. The study was terminated with the terminal sacrifice of F2 weanlings and F1 adult animals. Test diets containing the test material were offered continuously throughout the study.

Food consumption of the F0 and F1 parents was determined regularly during premating (once weekly) and additionally during gestation and lactation periods. In general, body weights of F0 and F1 parents were determined once weekly. However, during gestation and lactation females were weighed on days 0, 7, 14 and 20 of gestation, on the day of parturition, and on days 4, 7, 14 and 21 after delivery. Pups were weighed on the day after birth and on days 4, 7, 14 and 21 thereafter.

The parents' and pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances. Pups were sexed and monitored with respect to their development stages and their behavior in certain tests. Their viability was recorded. All pups were examined macroscopically at necropsy; if necessary, certain pups were additionally inspected for any organ/skeletal findings.

Blood and urine samples were taken from 12 F0 and 12 F1 males and females of each test group towards the end of the relevant study period for clinico-chemical examinations.

All F0 and F1 parental animals were assessed by gross pathology (including weight determinations of several organs) and subjected to an extensive histopathological examination, special attention being paid to the organs of the reproductive system.

The amount of test material administered (during premating (F0 and F1 parental animals)) was of an average of about 2 mg/kg body weight/day in the 20 ppm group, of about 10 mg/kg body weight/day in the 100 ppm group, and of about 50 mg/kg body weight/day in the 500 ppm group.

 

The following findings were obtained and assessed as test material-related:

500 ppm group (ca. 50 mg/kg body weight/day):

F0 parental animals

Clinical examinations: Nothing abnormal detected.

Clinical chemistry and urinalyses: Nothing abnormal detected.

Pathology: Increased absolute and relative kidney weights (both sexes).

F1a and F1b pups

Clinical examinations: Slightly increased number of F1a and F1b pups which died or were cannibalised (days 0 - 4 p.p.) and consequently reduced viability indices; marginal impairment of F1a pup body weight gain between days 7 - 14 p.p.

F1parental animals

Clinical examinations: Nothing abnormal detected.

Clinical chemistry and urinalyses: Nothing abnormal detected.

Pathology: Increased absolute and relative kidney weights (both sexes).

F2 pups

Clinical examinations: Increased number of pups which died or were cannibalised (days 0 - 4 p.p.) and consequently reduced viability index; lower mean pup body weights in comparison to the controls on days 14 - 21 p.p.: clear impairment of pup body weight gain between days 4 - 7, 7 - 14 and 4 - 21 p.p.; higher number of pups with delayed auditory canal opening.

 

100 ppm group (ca. 10 mg/kg body weight/day):

F0 parental animals: Nothing abnormal detected concerning clinical and clinico-chemical examinations and urinalyses.

Pathology: Increased relative kidney weights (males only).

F1a and F1b pups: Slightly increased number of F1b pups which died or were cannibalised (days 0 - 4 p.p.) and consequently reduced viability index.

F1parental animals: Nothing abnormal detected concerning clinical and clinico-chemical examinations and urinalyses.

Pathology: Increased absolute (males only) and relative (both sexes) kidney weights.

F2 pups: Nothing abnormal detected.

 

20 ppm groups (ca. 2.0 mg/kg body weight/day):

F0 parental animals: Nothing abnormal detected concerning clinical and clinico-chemical examinations, urinalyses and pathology

F1a and F1b pups: Nothing abnormal detected

F1 parental animals: Nothing abnormal detected concerning clinical and clinico-chemical examinations, urinalyses and pathology

F2 pups: Nothing abnormal detected.

 

It can be said in conclusion that the dietary administration of the test material to rats in doses of 500 ppm (ca. 50 mg/kg body weight/day) caused signs of systemic toxicity in the parental animals of both generations and their progeny. At this dose level, however, no indications for a test material-induced impairment of the reproductive function were present.

100 ppm (ca. 10 mg/kg body weight/day) were still marginally toxic, but there were no indications of a disturbed reproductive function of the F0 or F1 parents.

20 ppm (ca. 2 mg/kg body weight/day) were tolerated by the parental animals of both generations and by all litters (F1a, F1b and F2) without any test material-related adverse effects.

Under the conditions of the study, the NOAEL for reproductive effects was 500 ppm. (equivalent to ca. 40 mg/kg bw/day).

 The NOAEL for systemic toxicity of the test material was 100 ppm for F0 females, F1 and F2 pups, and 20 ppm for F0 males, for males and females of the P1 parental generation and the F1b pups.

Reference 4

Endpoint:

two-generation reproductive toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please see the read-across justification report in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

NOAEL

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Dose descriptor:

NOAEL

Effect level:

100 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

organ weights and organ / body weight ratios

Dose descriptor:

NOAEL

Effect level:

20 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

Critical effects observed:

yes

Lowest effective dose / conc.:

20 ppm

System:

urinary

Organ:

kidney

Treatment related:

not specified

Dose response relationship:

yes

Relevant for humans:

not specified

Dose descriptor:

NOAEL

Effect level:

20 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Dose descriptor:

NOAEL

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Critical effects observed:

no

Dose descriptor:

NOAEL

Generation:

F1a

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

Dose descriptor:

NOAEL

Generation:

F1b

Effect level:

20 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

Critical effects observed:

no

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

other: Higher number of pups with delayed auditory canal opening.

Critical effects observed:

no

Reproductive effects observed:

no

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

40 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Two generation study on the racemate and a one-generation study on the isomer, both conducted according to OECD Test Guidlines and in compliance with GLP.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

One-Generation Reproductive Toxicity: Clode (2003)

The reproductive toxicity of the test material was assessed according to OECD test Guideline 415 and in compliance with GLP.

The objective of the study was to provide a preliminary evaluation of the test material on the reproduction toxicity in the rat, and to define a Maximum Tolerated Dose (MTD) for a possible subsequent 2-generation study. As the compound was known to show a relatively steep dose relationship, dietary inclusion rates were amended during lactation to take account of the increased maternal food consumption at that time.

Groups of 12 male and 12 female rats were given the test material by dietary administration at nominal concentrations of 0 (control), 500, 800 and 1 200 ppm. Both sexes were treated for 10 weeks before pairing on a one to one basis within each group. The females continued with this treatment throughout gestation and then were allowed to litter and rear their offspring to weaning. During the lactation period, the females were given diets containing nominal concentrations of 0 (control), 300, 530 and 790 ppm the test material. Males were treated until evaluation of the females had been completed. Ten animals of each sex were randomly selected from each group to form the filial (F1) generation. These animals received the test material at the original concentrations for 4 weeks post-weaning.

Analysis of samples from the formulations prepared in Weeks 1, 11, 17 and 22 of the study showed that they were suitable for administration to the study animals.

P generation:

All animals survived to the scheduled kill.

There were no clinical observations that were related to treatment. Males in the high dose group gained less weight than the controls during the first 5 weeks of treatment. There was a dose-related reduction in female body weight gain during the pre-pairing period and gestation. Females in the high dose group gained less weight than the controls during the last week of the lactation period. Group mean food intake was lower than control in the intermediate and high dose group males and in the high dose females during the pre-pairing period. During gestation and lactation, females in the high dose group ate significantly less than the controls.

Mating performance and fertility were unaffected by treatment.

Mean duration of gestation was similar in all groups. In the high dose group, there was a reduction in mean number of implantation sites.

Pup viability was unaffected by treatment. Mean pup weight gain was slightly lower in the high dose group compared to control.

F1 generation:

Group mean body weight gain and food intake of the males and females in the high dose group were lower than control over the 4 week treatment period.

 

Under the conditions of the study dietary administration of 1 200 ppm of the test material to adult male and female rats for 10 weeks prior to mating, 790 ppm to the female rats during the lactation period, and again giving 1 200 ppm to the F1 generation for 4 weeks, produced slight adult and pup toxicity in terms of lower body weight gains and food intakes. At these concentrations there were fewer implantation sites compared to control.

At 500 and 800 ppm (reduced to 300 and 530 ppm during lactation), there were marginal effects on adult and pup weight gain and food intake and slightly lower numbers of implantation sites compared to control.

It is considered that reduction of dietary concentration by approximately one third during the lactation period in a 2-generation study, would allow demonstration of a maternal MTD without inducing excessive effects in the offspring.

Two-Generation Reproductive Toxicity: Hellwig (1992)

The reproductive toxicity of the test material was assessed according to OECD Test Guideline 416, JMAFF, EPA/FIFRA and European Commission guidelines and in compliance with GLP. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was administered to groups of 25 male and 25 female immature rats (F0 parental generation) as a constant homogeneous addition to the food in different dose levels (0, 20, 100 or 500 ppm). At least 70 days after the beginning of treatment, F0 animals were mated to produce a first litter (F1a) and subsequently re-mated to produce a second litter (F1b retained only until weaning). Groups of 25 males and 25 females selected from F1a pups as F1 parental generation were offered diets containing 0, 20, 100 and 500 ppm of the test material post weaning, and the breeding program was repeated to produce F2 litter. The study was terminated with the terminal sacrifice of F2 weanlings and F1 adult animals. Test diets containing the test material were offered continuously throughout the study.

Food consumption of the F0 and F1 parents was determined regularly during premating (once weekly) and additionally during gestation and lactation periods. In general, body weights of F0 and F1 parents were determined once weekly. However, during gestation and lactation females were weighed on days 0, 7, 14 and 20 of gestation, on the day of parturition, and on days 4, 7, 14 and 21 after delivery. Pups were weighed on the day after birth and on days 4, 7, 14 and 21 thereafter.

The parents' and pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances. Pups were sexed and monitored with respect to their development stages and their behavior in certain tests. Their viability was recorded. All pups were examined macroscopically at necropsy; if necessary, certain pups were additionally inspected for any organ/skeletal findings.

Blood and urine samples were taken from 12 F0 and 12 F1 males and females of each test group towards the end of the relevant study period for clinico-chemical examinations.

All F0 and F1 parental animals were assessed by gross pathology (including weight determinations of several organs) and subjected to an extensive histopathological examination, special attention being paid to the organs of the reproductive system.

The amount of test material administered (during premating (F0 and F1 parental animals)) was of an average of about 2 mg/kg body weight/day in the 20 ppm group, of about 10 mg/kg body weight/day in the 100 ppm group, and of about 50 mg/kg body weight/day in the 500 ppm group.

The following findings were obtained and assessed as test material-related:

500 ppm group (ca. 50 mg/kg body weight/day):

F0 parental animals

Clinical examinations: Nothing abnormal detected.

Clinical chemistry and urinalyses: Nothing abnormal detected.

Pathology: Increased absolute and relative kidney weights (both sexes).

F1a and F1b pups

Clinical examinations: Slightly increased number of F1a and F1b pups which died or were cannibalised (days 0 - 4 p.p.) and consequently reduced viability indices; marginal impairment of F1a pup body weight gain between days 7 - 14 p.p.

F1parental animals

Clinical examinations: Nothing abnormal detected.

Clinical chemistry and urinalyses: Nothing abnormal detected.

Pathology: Increased absolute and relative kidney weights (both sexes).

F2 pups

Clinical examinations: Increased number of pups which died or were cannibalised (days 0 - 4 p.p.) and consequently reduced viability index; lower mean pup body weights in comparison to the controls on days 14 - 21 p.p.: clear impairment of pup body weight gain between days 4 - 7, 7 - 14 and 4 - 21 p.p.; higher number of pups with delayed auditory canal opening.

100 ppm group (ca. 10 mg/kg body weight/day):

F0 parental animals: Nothing abnormal detected concerning clinical and clinico-chemical examinations and urinalyses.

Pathology: Increased relative kidney weights (males only).

F1a and F1b pups: Slightly increased number of F1b pups which died or were cannibalised (days 0 - 4 p.p.) and consequently reduced viability index.

F1parental animals: Nothing abnormal detected concerning clinical and clinico-chemical examinations and urinalyses.

Pathology: Increased absolute (males only) and relative (both sexes) kidney weights.

F2 pups: Nothing abnormal detected.

20 ppm groups (ca. 2.0 mg/kg body weight/day):

F0 parental animals: Nothing abnormal detected concerning clinical and clinico-chemical examinations, urinalyses and pathology

F1a and F1b pups: Nothing abnormal detected

F1 parental animals: Nothing abnormal detected concerning clinical and clinico-chemical examinations, urinalyses and pathology

F2 pups: Nothing abnormal detected.

It can be said in conclusion that the dietary administration of the test material to rats in doses of 500 ppm (ca. 50 mg/kg body weight/day) caused signs of systemic toxicity in the parental animals of both generations and their progeny. At this dose level, however, no indications for a test material-induced impairment of the reproductive function were present.

100 ppm (ca. 10 mg/kg body weight/day) were still marginally toxic, but there were no indications of a disturbed reproductive function of the F0 or F1 parents.

20 ppm (ca. 2 mg/kg body weight/day) were tolerated by the parental animals of both generations and by all litters (F1a, F1b and F2) without any test material-related adverse effects.

Under the conditions of the study, the NOAEL for reproductive effects was 500 ppm. The NOAEL for systemic toxicity of the test material was 100 ppm for F0 females, F1 and F2 pups, and 20 ppm for F0 males, for males and females of the P1 parental generation and the F1b pups.

Effects on developmental toxicity

Description of key information

Developmental Toxicity in Rats: Hellwig (1993)

Under the conditions of the study the no observed adverse effect level (NOAEL) on maternal and foetal rats is 50 mg/kg body weight/day.

Developmental Toxicity in Rabbits: Hellwig (1993)

Under the conditions of the study the no observed adverse effect level (NOAEL) on the maternal and foetal rabbits is 50 mg/kg body weight/day.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 July 1991 to 31 July 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

Version / remarks:

1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: "Commission Directive 87/302/EEC of Nov. 18, 1987 adapting to technical progress for the ninth time; Council Directive 67/548/EEC, pp. 24 - 26".

Version / remarks:

1988

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Chbb:THOM (SPF)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: The female rats were about 53 or 56 days old when supplied. At the beginning of the study (day 0, detection of sperm), the rats were 61 or 66 days old.
- Weight at study initiation: Their mean weight was approx. 222.8 g.
- Housing: During the study period, the rats were housed singly in type DK III stainless steel wire mesh cages.
- Diet: Ad libitum throughout the study (from the day of supply to the day of necropsy).
- Water: Ad libitum throughout the study (from the day of supply to the day of necropsy).
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70 %
- Photoperiod (hrs dark / hrs light): The day/night rhythm was 12 hours (12 hours light from 6.00 to 18.00 hours and 12 hours darkness from 18.00 to 6.00 hours).

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5 % aqueous CMC solution.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Each day the test material suspensions were freshly prepared shortly before the test material was administered. For the preparation of the suspensions, an appropriate amount of the test material was weighed and subsequently suspended in a 0.5 % aqueous carboxymethyl cellulose solution using a high speed sonicator. A magnetic stirrer was used to keep the suspensions homogeneous during treatment of the animals.
Due to technical reasons, the study was carried out in 2 sections. Each dose group was represented in each section. A treatment interval of 2 days elapsed before the next section.

VEHICLE
- Concentration in vehicle: 0, 200, 500 and 1 000 mg/100 mL.
- Amount of vehicle: 10 mL/kg The calculation of the volume administered was based on the individual body weight determined at the beginning of the administration period (day 6 p.c.).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical determinations of the purity and the homogeneity of the test material itself were carried out before the beginning of the study (method: HPLC).
A reanalysis of the test material for verification of the stability will be carried out after completion of the current series of studies with this batch of test material.

Analyses of the suspensions of test material:
Analytical verifications of the stability of the test material suspensions up to 24 hours were carried out before the beginning of the study.
The stability of the test material suspensions was checked in the range-finding study in a comparable batch. Samples of the test material suspensions were sent to the analytical laboratories twice during the study period for verification of the concentrations. The samples which were sent for the first concentration control analysis toward the beginning of the administration period were also used to verify the homogeneity for the samples of the low and the high concentrations (20 and 100 mg/kg bw/day). Six samples (2 from the top, middle and bottom in each case) were taken for each of these concentrations from the beaker with a magnetic stirrer running. The test material suspensions were analysed by HPLC.

Measurement Method
Reverse-phase-HPLC with UV-detection.
The concentration control analyses yielded mean concentrations of 94.4 % and 99.2 % for the 500 mg/100 mL, 96.4 % for the 200 mg/100 mL and 103.4 % for the 1 000 mg/100 mL samples.
The analyses of the homogeneity yielded mean concentrations and standard deviations of 102.8 % (± 5.15 %) and 92.4 % (± 2.68 % for the 200 and 1 000 mg/100 mL samples respectively.
The concentration control analyses confirm the theoretical values, the CMC-suspension is mixed homogeneousely.

Results of analyses of the test material:
The homogeneity of the test material was proven. The content of active ingredient was determined twice before the beginning of the study. The results of these determinations showed a purity of 92.7 % and 92.2 %.

Results of analyses of the suspensions of test material:
The stability of the test material suspensions over a period of up to 24 hours at room temperature was demonstrated.
The results of the analyses of the suspensions of test material confirmed the correctness of the prepared concentrations and the homogeneous distribution of the test material in the vehicle.

Details on mating procedure:

- Impregnation procedure: Cohoused
If cohoused:
- M/F ratio per cage: Four untreated female rats were mated with one untreated fertile male animal of the same breed.
- Verification of same strain and source of both sexes: Animals were of the same breed.
- Proof of pregnancy: If sperm were detected microscopically in the vaginal smear the animals were considered to be fertilised. This day was designated "day 0" (beginning of the study) and the following day "day 1" post coitum (p.c.).

Duration of treatment / exposure:

Once a day during the period of major organogenesis (day 6 to day 15 p.c.) always at approx. the same time of day (in the morning).

Frequency of treatment:

Daily

Duration of test:

Day 6 to day 15 p.c.

Dose / conc.:

20 mg/kg bw/day (actual dose received)

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25 females per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The selection of doses for the present examination was based on the results of several preceding studies, in particular a range-finding study.
In a range-finding study, the test material was administered to pregnant rats (10/dose) by gavage from day 6 to day 15 post coitum at doses of 0, 50, 100 and 150 mg/kg bw/day. For a clear assessment of test material-related effects on the dams, the animals were sacrificed on day 16 p.c. Just before sacrifice, blood samples were taken for clinico-chemical and haematological examinations. Furthermore, liver and kidneys were weighed. Due to the sacrifice of the dams on day 16 p.c., only limited information on embryonic and foetal toxicity could be obtained.
150 mg/kg bw/day:
- clearly reduced food consumption between days 6 - 13 p.c.;
- statistically significantly lower body weights than in the control group between days 13 - 16 p.c.;
- body weight loss between days 6 - 8 p.c., reduced body weight gains on the following days;
- drastically reduced gravid uterine weights;
- drastically reduced corrected body weight gain;
- statistically significant reduction of erythrocytes, haemoglobin, haematocrit and platelets;
- significant increase in alanine aminotransferase and alkaline phosphatase;
- increased chloride, creatinine, urea and glucose values;
- decreased total protein, globulin, triglyceride and cholesterol values statistically;
- significantly increased absolute and relative liver weights (13 or 20 % higher than the relevant control values);
- statistically significant increase of the relative kidney weights (6 %)
- clearly increased post-implantation loss due to the increase a number of early and late resorptions;
- reduced number of live foetuses/dam;
- decreased mean foetal body weights.

100 mg/kg bw/day:
- clearly reduced food consumption between days 6 - 10 p.c.;
- reduced body weight gains on days 6 - 8 p.c.;
- marginally reduced corrected body weight gain;
- statistically significant reduction of erythrocytes, haemoglobin, haematocrit and platelets;
- increase in alanine aminotransferase (trend) and statistically significantly increased alkaline phosphatase values;
- increased glucose values;
- decreased total protein, globulin, triglyceride and cholesterol values;
- increased absolute (trend) and statistically significantly increased relative liver weights (10 or 13 % higher than the relevant control values);
- slightly increased post-implantation loss.

50 mg/kg bw/day:
- marginally reduced food consumption between days 6 - 8 p.c.;
- marginally reduced corrected body weight gain [questionable substance effect];
- statistically significant reduction of haemoglobin and haematocrit values;
- statistically significantly decreased cholesterol values.

Taking into consideration the results of this study with administration of the test material the following doses were fixed for the full-scale prenatal toxicity study in rats:
20 mg/kg bw: As the expected no observed adverse effect level (NOAEL).
50 mg/kg bw: As a dose which might be a minimal toxic effect level for dams and/or foetuses.
100 mg/kg bw: As the dose level with maternally toxic effects at which findings in foetuses may also be obtained.

- Rationale for animal assignment: On day 0, the animals were assigned to the different test groups according to a randomisation plan.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check was made twice a day on working days or once a day (Saturday, Sunday or on public holidays) (days 0 - 20 p.c.).
- Cage side observations checked: Mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were examined for clinical symptoms at least once a day, or more often when clinical signs of toxicity were elicited (days 0 - 20 p.c.).

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on days 0, 1, 3, 6, 8, 10, 13, 15, 17 and 20 p.c. The body weight change of the animals was calculated from these results.
Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on day 20 p.c. minus weight of the uterus before it was opened minus body weight on day 6 p.c.).

FOOD CONSUMPTION: Yes
- With the exception of day 0, the consumption of food was determined on the same days as was body weight.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes. On day 20 p.c., all females were sacrificed in a randomised order and examined macroscopically. The foetuses were dissected from the uterus and further investigated with different methods.
- Sacrifice on gestation day: On day 20 p.c., the dams were sacrificed in randomised order by cervical dislocation and the foetuses dissected from the uterus. After the dams had been sacrificed, they were necropsied and assessed by gross pathology. The uterus and the ovaries were removed.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes, including distribution of implantation sites.
- Number of early resorptions: Yes, early resorptions (only decidual or placental tissues visible or according to Salewski from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single-horn pregnancy).
- Number of late resorptions: Yes, embryonic or foetal tissue in addition to placental tissue visible.

Fetal examinations:

- External examinations: Yes: At necropsy each foetus was weighed, sexed and examined macroscopically for any external findings. The sex was determined by observing the distance between the anus and the base of the genital tubercle and was later confirmed in all foetuses fixed in Bouin's solution by internal examination. If there were discrepancies between the "external" and the "internal" sex of a foetus, the foetus was finally sexed according to the appearance of its gonads.
Furthermore, the viability of the foetuses and the condition of the placentae, the umbilical cords, the foetal membranes and fluids were examined. Individual placental weights were recorded.
After these examinations, approximately one half of the foetuses per dam was placed in ethyl alcohol and the other half was placed in BOUIN's solution for fixation and further evaluation.
- Soft tissue examinations: Yes: After fixation in Bouin's solution, approximately one half of the foetuses of the dams of all groups was examined for any findings in the organs according to the method of Barrow and Taylor. After these examinations the relevant foetuses were discarded.
- Skeletal examinations: Yes: After fixation in ethyl alcohol, the skeletons of approximately one half of the foetuses were stained according to a modified method of Dawson. Thereafter, the skeletons of these foetuses were examined under a stereomicroscope. After these examinations the relevant foetuses were retained by litter.
- Head examinations: Not specified.

Evaluation criteria for assessing skeletons and organs of the foetuses:
There are differing opinions on the classification and assessment of changes in foetuses. Müntefering differentiates between malformations, which he defines as "severe morphological defects outside the range of variation of the species", and macroscopic/microscopic anomalies, which he defines as "slighter morphological deviations from normal". If these changes occur, they are regarded by Müntefering as a teratogenic effect. However, transition from variation to malformation is fluid, and the term "slighter morphological deviation" is not defined. Neubert, on the other hand, tends to describe a change as a morphological abnormality, anomaly or functional anomaly instead of malformation since malformation refers primarily to gross-pathological changes.
In the present investigations the following terms (definitions) were used for describing a change:
- Malformations (concerning external, soft tissue and skeletal observations): Rare and/or probably lethal changes were classified as malformations (e.g. exencephaly, atresia ani, hernia umbilicalis).
- Variations (concerning external, soft tissue and skeletal observations): Changes which occur regularly also in control groups and have generally no adverse effect on survival were regarded as variations (e.g. dilated renal pelvis).
- Retardations (concerning skeletal observations only): Delays in skeletal development compared with the norm at the time of the examination were considered to be retardations (e.g. sternebra(e) not ossified).
- Unclassified observations (concerning external and soft tissue observations, only): External or soft tissue observations, which could not be classified as malformations or variations (e.g. blood coagulum around placenta or urinary bladder).

Statistics:

The data were evaluated statistically using the computer systems of the Department of Toxicology of BASF Aktiengesellschaft.

Examinations of dams and foetuses:
Dunnet's Test was used for statistical evaluation of food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), weight of the uterus before it was opened, weight of foetuses, weight of placentae, corpora lutea, implantations, pre- and post-implantation losses, resorptions and live foetuses.
Fisher's Exact Test was used for statistical evaluation of conception rate, mortality (of the dams) and all foetal findings.

Indices:

Calculations of conception rate and pre- and post-implantation losses were carried out.
The conception rate (in %) was calculated according to the following formula:

(number of pregnant animals / number of fertilised animals) x 100

The pre-implantation loss (in %) was calculated* according to the following formula:

((number of corpora lutea - number of implantations) / number of corpora lutea) x 100

The post- implantation loss (in %) was calculated* from the following formula:

((number of implantations - number of live foetuses) / number of implantations) x 100

* Calculation on the basis of each individual pregnant animal with scheduled sacrifice.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no abnormal clinical findings in any dam of any one group.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no mortalities in any of the groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of the 100 mg/kg rats were slightly, but statistically significantly lower than the relevant control value on day 8 p.c. At the beginning of the treatment period (days 6 – 8 p.c.) the high dose dams (100 mg/kg bw/ day) lost some body weight; in the subsequent time, however, weight gain of the high dose dams sometimes reached or even exceeded (especially on days 8 - 10 p.c.) control values. If the body weight gain of the high dose dams during the whole treatment period, however, is calculated, these dams gained about 18 % less than the control dams.
Body weights and body weight gains of the dams of low and intermediate dose dams (20 mg/kg bw/day and 50 mg/kg bw/day) were similar to those of the controls. All observed differences between these groups are without any biological relevance.
The corrected body weight gain (terminal body weight on day 20 p.c. minus weight of the uterus before it was opened minus body weight on day 6 p.c.) was statistically significantly reduced in high dose dams (100 mg/kg bw/day), which has to be assessed as a test material-related effect. All other differences between the groups are without any biological relevance.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption of the intermediate and high dose dams was statistically significantly reduced at the beginning of the treatment period (50 mg/kg group: days 6 - 8 p.c.; 100 mg/kg group: days 6 - 10 p.c.). The reduction in food intake (up to about 22 % less food consumption than the controls) of the high dose dams has to be assessed as being clearly related to the test material administration because body weights, body weight gain and corrected body weight gain of these animals were also impaired. The marginally reduced food consumption of the 50 mg/kg dams, which consumed up to about 9 % less food than the controls, however, is assessed as being a spontaneous effect, because it was not accompanied by statistically significant impairments of body weights/weight gains.
The food consumption of the low dose dams (20 mg/kg bw/day) did not show any differences of biological relevance if compared to the controls.
During the post treatment period (days 15 - 20 p.c.) food consumption of the high dose dams reached control values.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The uterus weights of the animals were not influenced by the administration of the test material. The differences between the groups are without biological relevance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test material-related observations at necropsy in any of the dams. Oedema of the lungs were recorded for one control, one low dose and two high dose dams. A haemorrhagic liver was recorded for one control rat (No. 17) and one low dose rat (No. 31) showed a hydrometra; neither of these animals became pregnant.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

Only pregnant dams were used for the calculations of mean maternal food consumption, body weight and body weight change.
Only pregnant dams with scheduled sacrifice (day 20 p.c.) were taken for the calculation of mean gravid uterine weights, mean net maternal body weight change (corrected body weight gain) and summary of reproduction data.
In this study the following females were partially or totally excluded from the above mentioned calculations:
Control dams (0 mg/kg bw/day): Female No. 17 - not pregnant.
Low dose dams (20 mg/kg bw/day): Females Nos. 30, 31, 42, 43 and 47 - not pregnant.
Intermediate dose dams (50 mg/kg bw/day): Females Nos. 71 and 72 - not pregnant.
High dose dams (100 mg/kg bw/day): Females Nos. 86, 87, 88, 89 and 90 - not pregnant.

Number of abortions:

not specified

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no test material-related and/or statistically significant differences of biological relevance between the groups in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the post-implantation losses. The differences evident are considered to be incidental and within the normal range of deviations for animals of this strain and age.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There were no test material-related and/or statistically significant differences of biological relevance between the groups in the number of resorptions and viable foetuses. The differences evident are considered to be incidental and within the normal range of deviations for animals of this strain and age.

Early or late resorptions:

no effects observed

Description (incidence and severity):

There were no test material-related and/or statistically significant differences of biological relevance between the groups in the number of resorptions. The differences evident are considered to be incidental and within the normal range of deviations for animals of this strain and age.

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test material-related and/or statistically significant differences of biological relevance between the groups in the number of viable foetuses. The differences evident are considered to be incidental and within the normal range of deviations for animals of this strain and age. This includes the incidental, relatively high number of live foetuses in the 100 mg/kg group, which is clearly higher than the corresponding control value (13.9 versus 12.8 live foetuses/dam) but is still fully within the historical control range (11.1 - 14.9 live foetuses/ dam). The high number of live foetuses in the 100 mg/kg group, however, is the reason for the marginally, but statistically significantly lower mean foetal body weight and the increased number of skeletal retardations in this group.

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The conception rate varied between 96 % (control group) and 80 % (20 and 100 mg/kg groups). There were no test material-related and/or statistically significant differences of biological relevance between the groups in conception rate. The differences evident are considered to be incidental and within the normal range of deviations for animals of this strain and age. This includes the incidental, relatively high number of live foetuses in the 100 mg/kg group, which is clearly higher than the corresponding control value (13.9 versus 12.8 live foetuses/dam) but is still fully within the historical control range (11.1 - 14.9 live foetuses/ dam).

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The differences evident are considered to be incidental and within the normal range of deviations for animals of this strain and age. This includes the incidental, relatively high number of live foetuses in the 100 mg/kg group, which is clearly higher than the corresponding control value (13.9 versus 12.8 live foetuses/dam) but is still fully within the historical control range (11.1 - 14.9 live foetuses/ dam). The high number of live foetuses in the 100 mg/kg group, however, is the reason for the marginally, but statistically significantly lower mean foetal body weight and the increased number of skeletal retardations in this group.

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test material-related and/or statistically significant differences of biological relevance between the groups in the number of viable foetuses. The differences evident are considered to be incidental and within the normal range of deviations for animals of this strain and age. This includes the incidental, relatively high number of live foetuses in the 100 mg/kg group, which is clearly higher than the corresponding control value (13.9 versus 12.8 live foetuses/dam) but is still fully within the historical control range (11.1 - 14.9 live foetuses/ dam).

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the foetuses in the treatment groups (20, 50 and 100 mg/kg bw/day) was comparable with the control foetuses. The differences observed in comparison to the control are without any biological relevance.

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

Weight of foetuses: The mean foetal weights were not influenced by the test material administration. All values are within the range of biological variation. This includes the marginally, but statistically significantly lower foetal body weight in the 100 mg/kg group, which is caused by the increased number of live foetuses in this group.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

An external malformation was recorded for one control foetus, only. For this foetus, an anasarca was noted. This malformation is also present in the historical control data. The external examination of the foetuses revealed no variations in any group. One so-called unclassified observation (placentae fused) was recorded for one foetus of test group 2 and one foetus of test group 3 (50 or 100 mg/kg bw/day).

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Various malformations of the sternum (sternebra(e) bipartite, ossification centers dislocated) and/or the vertebral column (thoracic vertebral body/bodies dumbbell-shaped (asym.) or bipartite (asym.), lumbar vertebral body absent) were seen in 10 out of 158 (~ 6.3 %) foetuses (in 8 out of 24 litters (~ 33 %)) of the control, in 8 out of 141 (~ 5.7 %) foetuses (in 6 out of 20 litters (~ 30 %)) of the 20 mg/kg group, in 7 out of 155 (~ 4.5 %) foetuses (in 6 out of 23 litters (~ 26 %)) of the 50 mg/kg group and in 4 out of 144 (~ 2.8 %) foetuses (in 3 out of 20 litters (~ 15 %)) of the 100 mg/kg group. These differences were not statistically significant and did not show any dose-response relationship.
The variations elicited were related to the ribs (shortened 13th, accessory 14th ribs, rudimentary cervical or wavy ribs) and the sternum (sternebra(e) of irregular shape, bipartite, or accessory sternebra). Most of the skeletal variations recorded appeared without a clear dose-response relationship (e.g. sternebra(e) of irregular shape, sternebra(e) bipartite, 13th rib(s) shortened), can be found in a similar frequency in the historical control data and/or the differences between the groups are without biological relevance; the statistically significantly increased number of 100 mg/kg foetuses, however, which showed rudimentary cervical rib(s), has to be related to the oral administration of the test material to the dams.
In all groups signs of retardations (incomplete or missing ossification of vertebral bodies/arches, sternebra(e), the skull and/or the hyoid bone) were found. In the 100 mg/kg group the number of foetuses with unossified sternebra(e) and the overall number of foetuses with skeletal retardations is statistically significantly increased. The foetal and litter incidence of "not ossified sternebra(e)" [17 % of the foetuses; 60 % of the litters] is just above the historical control range (3.4 - 16.0 % of the foetuses; 16.7 - 58.3 % of the litters). The increased number of high dose foetuses with retarded ossification, however, is not assessed as being test material-induced, but has to be related to the incidental, distinctly higher number of live foetuses/ dam in this test group in comparison to the control.
The foetal and litter incidences of skeletal retardations in test groups 1 and 2 (20 or 50 mg/kg body weight/day) do not show any statistically significant differences if compared to control values; the relevant values are fully in the range of the historical control.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The examination of the organs of the foetuses revealed no malformations in any of the groups. Variations (dilated renal pelvis and/or hydroureter) were detected in all groups without any statistically significant differences between the groups. All values are fully in the range of biological variation. No so-called unclassified observations (like blood coagulum around the placenta or urinary bladder) were recorded.

Other effects:

no effects observed

Description (incidence and severity):

Weight of placentae: The mean placental weights in the treatment groups (20, 50 or 100 mg/kg bw/day) were not influenced by the test material administration and were similar to the control values.

Key result

Dose descriptor:

NOAEL

Remarks:

(embryo-/foetotoxicity)

Effect level:

50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

skeletal malformations

Remarks on result:

other: No test material-induced teratogenic effects observed.

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: rib

Description (incidence and severity):

At 100 mg/kg bw/day marginal signs of embryo-/foetotoxicity in the form of an increased occurrence of foetuses with rudimentary cervical rib(s) were present, but no test material-induced teratogenic effects were seen up to and including the dose of 100 mg/kg bw/day.

Key result

Developmental effects observed:

no

Historical Control Data in Rats (Strain CHBB/THOM (SPF))

Skeletal Defect	Vehicle Controls N	Controls N	% of Total Malformations	% of Foetuses
Wavy ribs	6	22	9.78	0.04

 

Conclusions:

Under the conditions of the study the no observed adverse effect level (NOAEL) on maternal and foetal rats is 50 mg/kg bw/day.

Executive summary:

The test material was assessed for its prenatal toxicity in Wistar rats according to OECD Test Guideline 414 and in compliance with GLP.

The test material was administered as an aqueous suspension to 20 - 24 pregnant female rats/group by stomach tube at doses of 20, 50 and 100 mg/kg bw on day 6 through day 15 post coitum (p.c.). A standard dose volume of 10 mL/kg body weight was used. The control group was dosed with the vehicle only (0.5 % aqueous carboxymethyl cellulose solution).

Food consumption and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day.

On day 20 post coitum, all females were sacrificed and assessed by gross pathology. The foetuses were dissected from the uterus, sexed, weighed and further investigated for any external, soft tissue and/or skeletal findings. The following findings were obtained and assessed as test material-related:

High dose dams (100 mg/kg bw/day):

- Statistically significantly reduced food consumption at the beginning of treatment period (days 6 - 10 p.c.);

- Statistically significantly lower mean body weights than the controls on day 8 p.c.; slight body weight loss on days 6 - 8 p.c. and, if calculated for the whole treatment period, a weight gain of about 18 % less than that of controls;

- Reduced corrected body weight gain;

- Statistically significantly increased number of foetuses with a skeletal variation (rudimentary cervical rib(s)).

Intermediate dose dams (50 mg/kg bw/day):

- No test material-related effects on dams or foetuses.

Low dose dams (20 mg/kg bw/day):

- No test material-related effects on dams or foetuses.

Thus, under the conditions of this full-scale study, the test material caused some overt signs of maternal toxicity and marginal signs of embryo-/foetotoxicity at 100 mg/kg bw/day. No test material-induced teratogenic effects were observed up to and including the dose of 100 mg/kg bw/day. There were no test material-induced, adverse effects on dams or foetuses of the intermediate or low dose groups (50 or 20 mg/kg bw/day).

In the preceding range-finding study, however, which was carried out under comparable study conditions (e.g. same batch, same rat strain, same treatment schedule), massive signs of maternal toxicity in the form of clearly reduced food consumption, retarded body weight gain, impairment of several haematological and clinicochemical parameters and/or increased liver and kidney weights were found at doses of 100 and 150 mg/kg bw/day, whereas marginal maternally toxic effects were still present at 50 mg/kg bw/day.

Under the conditions of the study the no observed adverse effect level (NOAEL) on maternal and foetal rats is 50 mg/kg bw/day.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 July 1991 to 27 August 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

Version / remarks:

1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: "Commission Directive 87/302/EEC of Nov. 18, 1987 adapting to technical progress for the ninth time; Council Directive 67/548/EEC, pp. 24 - 26".

Version / remarks:

1988

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

Himalayan

Remarks:

Chbb:HM (outbred strain)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: At the beginning of the study (day 0) the does were between 21 and 26 weeks old.
- Weight at study initiation: The mean body weight was approx. 2 542 g.
- Housing: During the acclimatisation and the study periods, the rabbits were housed singly in type K 300/8 stainless steel wire mesh cages.
- Diet: Ad libitum throughout the study (from the day of supply to the day of necropsy).
- Water: Ad libitum throughout the study (from the day of supply to the day of necropsy).
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): Relative humidity of 30 - 70 %
- Photoperiod (hrs dark / hrs light): The day/night rhythm was 12 hours (12 hours light from 6.00 to 18.00 hours and 12 hours darkness from 18.00 to 6.00 hours).

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5 % aqueous CMC solution.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Each day the test material suspensions were freshly prepared shortly before the test material was administered. For the preparation of the suspensions, an appropriate amount of the test material was weighed and subsequently suspended in a 0.5 % aqueous carboxymethyl cellulose solution using a high speed sonicator (Ultra Turrax, JANKE & KUNKEL KG, FRG). A magnetic stirrer was used to keep the suspensions homogeneous during treatment of the animals.
Due to technical reasons the study was carried out in 3 sections. Each dose group was represented in each section. A treatment interval of 7 days elapsed before the next section.

VEHICLE
- Concentration in vehicle: 50, 200 or 500 mg/100 mL
- Amount of vehicle: The volume administered each day was 10 mL/kg body weight. The calculation of the volume administered was based on the individual body weight determined at the beginning of the administration period (day 7 p.i.).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

All analyses were carried out at the analytical laboratories of BASF Aktiengesellschaft.
Analyses of the Test Material:
Analytical determinations of the purity and the homogeneity of the test material itself were carried out before the beginning of the study (method: HPLC).
A reanalysis of the test material for verification of its stability will be carried out after completion of the current series of studies with this batch of test material.

Measurement Method
Reversed Phase-HPLC with UV-Detection
The analyses of homogeneity yielded mean concentrations and relative standard deviations of 97.6 % (± 2.7 %) and 96.2 % (± 6.4 %) for the 50 and 500 mg/100 mL samples. The 500 ppm-samples show a coefficient of variance of ± 6.4 %, the result is confirmed to CV = ± 7.0 % in a second series of analyses. The values of the relative standard variation are slightly higher than the analytical limit for a homogeneous mixture (CV= ± 5.0 %).
The concentration control analyses yielded mean concentrations of 95.9 % (samples from 24.07.1991) and 101 % (samples from 15.08.91) of the nominal concentration for the 200 mg/100 mL samples.
The analyses of the samples with nominal concentrations of 50 and 500 mg/100 mL yielded values of 101 % and 89.8 % of theoretical values (samples from 15.08.91) respectively. The results confirm the theoretical concentrations of the samples.

Analyses of the Test Material
The homogeneity of the test material was proven. The content of active ingredient was determined twice before the beginning of the study. The results of these determinations showed a purity of 92.7 % and 92.2 %.

Analyses of the Suspensions of the Test Material
The stability of the test material suspensions over a period of up to 24 hours at room temperature was demonstrated.
The results of the analyses of the test material suspensions confirmed the correctness of the prepared concentrations and the homogeneous distribution of the test material in the vehicle.

Analyses of the Suspensions of Test Material:
Analytical verifications of the stability of suspensions of the test material for a period of at least 24 hours at room temperature were carried out for a similar batch in a prenatal range-finding study in rats.
Samples of the test material suspensions were sent to the analytical laboratories twice during the study period for verification of the concentrations. The samples which were sent for the first concentration control analysis toward the beginning of the administration period were also used to verify the homogeneity for the samples of the low and the high concentrations (5 and 50 mg/kg bw/day). Six samples (two from the top, middle and bottom in each case) were taken for each of these concentrations from the beaker with a magnetic stirrer running. Moreover retain samples of the test material suspension (test group 3, 50 mg/kg bw/day) were sent to the analytical laboratories (for additional verification of the analytical results). The test material suspensions were analysed by HPLC.

Details on mating procedure:

- Impregnation procedure: Artificial insemination
This implied that 0.2 mL of a synthetic hormone which releases LH and FSH from the anterior pituitary lobe (Receptal®, trademark of HOECHST AG, Frankfurt) were injected intramuscularly to the female rabbits about 1 hour before insemination. The pooled ejaculate samples used for the artificial insemination were derived from male Himalayan rabbits of the same breed as the females.
The male donors were kept under conditions (air conditioning, diet, water) comparable to those of the females participating in this study.
The day of insemination was designated as day 0 (beginning of the study) and the following day as day 1 post insemination (p.i.).

Duration of treatment / exposure:

The test material was administered to the animals orally (by gavage) once a day during the period of major organogenesis (day 7 to day 19 p.i.) always at approx. the same time of day (in the morning).

Frequency of treatment:

Once daily

Duration of test:

29 days

Dose / conc.:

5 mg/kg bw/day

Dose / conc.:

20 mg/kg bw/day

Dose / conc.:

50 mg/kg bw/day

No. of animals per sex per dose:

15 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The selection of doses for the present examination was based on the results of several studies.
In a range-finding study carried out shortly before the start of the full-scale study, the test material was administered to pregnant rabbits (5/dose) by gavage from day 7 to day 19 post insemination at doses of 0, 40, 80 and 120 mg/kg bw/day. For a clear assessment of test material-related effects on the dams, the animals were sacrificed on day 20 p.i. Just before sacrifice, blood samples were taken for clinicochemical and haematological examinations. Furthermore, liver and kidneys were weighed. Due to the sacrifice of the dams on day 20 p.i. only limited information on embryonic and foetal toxicity could be obtained.
The following findings were obtained and assessed as possibly being test material-related:

120 mg/kg bw/day:
- drastically reduced food consumption between days 7 - 19 p.i.;
- body weight loss during the whole treatment period (days 7 - 19 p.i.);
- drastically reduced corrected body weight gain;
- intercurrent death of two dams, which showed severe adverse clinical symptoms (like abdominal or lateral position, salivation, no defecation) on the days before death; furthermore, for two dams with scheduled sacrifice no defecation was recorded at the end of the treatment period (day 19 p.i.);
- statistically significant reduction of erythrocytes, haemoglobin and haematocrit values;
- increased mean cell volume and mean corpuscular haemoglobin;
- increased polymorphonuclear neutrophils and leucocytes;
- increased polychromasia, anisocytosis, poikilocytosis, microcytosis, macrocytosis and normoblasts;
- increased creatinine, triglyceride, cholesterol, total bilirubin and urea values;
- decreased sodium, potassium and chloride values;
- statistically significant increase of the relative kidney weights (20 %);
- clearly increased post-implantation loss due to the increase number of early and late resorptions;
- reduced number of live foetuses/dam;
- decreased mean foetal body weights.

80 mg/kg bw/day:
- clearly reduced food consumption during the treatment period (days 7 - 19 p.i.);
- body weight loss during the whole treatment period (days 7 - 19 p.i.);
- reduced corrected body weight gain;
- increased creatinine values;
- decreased mean foetal body weights (without being statistically significant).

40 mg/kg bw/day:
- marginally reduced food consumption between days 7 - 10 p.i.;
- body weight loss during the whole treatment period (days 7 - 19 p.i.);
- reduced corrected body weight gain;
- increased post-implantation loss [questionable effect due to missing dose-response relationship];
- reduced number of live foetuses/dam [questionable effect due to missing dose-response relationship];
- decreased mean foetal body weights.

Taking into consideration the results of the above study with administration of the test material, the following doses were fixed for the full-scale prenatal toxicity study in rabbits with the test material:
5 mg/kg bw: As the expected no observed adverse effect level (NOAEL).
20 mg/kg bw: As a dose which might be a minimal toxic effect level for dams and/or foetuses.
50 mg/kg bw: As the dose level with maternally toxic effects at which findings in foetuses may also be obtained.

- Rationale for animal assignment: During the acclimatisation period the animals were assigned to the different test groups according to a randomisation plan and on the basis of their body weights.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check was made twice a day on working days or once a day (Saturday, Sunday or on public holidays) (days 0 - 29 p.i.).
- Cage side observations checked: Mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were examined for clinical symptoms at least once a day, or more often when clinical signs of toxicity were elicited (days 0 - 29 p.i.).

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on days 0, 2, 4, 7, 9, 11, 14, 16, 19, 21, 23, 25 and 29 p.i. The body weight change of the animals was calculated from these results.
Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on day 29 p.i. minus weight of the uterus before it was opened minus body weight on day 7 p.i.).

FOOD CONSUMPTION: Yes
- The consumption of food was determined daily during the entire study period.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes.
- Sacrifice on gestation day: On day 29 p.i., all surviving females were sacrificed in randomised order and examined macroscopically. The foetuses were dissected from the uterus and further investigated with different methods.
- Organs examined: On day 29 p.i., the surviving dams were sacrificed in randomised order by intravenous injection of a pentobarbital and the foetuses were dissected from the uterus. Dams which died intercurrently as well as the contents of the uterus from these animals were investigated, if possible in the same way as at terminal sacrifice (exception: uterus weight). After the dams had been sacrificed, they were necropsied and assessed by gross pathology. The uterus and the ovaries were removed.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes. Weight of uterus before it was opened.
- Number of corpora lutea: Yes
- Number of implantations: Yes. Number and distribution of implantation sites classified as live foetuses.
- Number of early resorptions: Yes. Only decidual or placental tissues visible or according to Salewski from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single-horn pregnancy.
- Number of late resorptions: Yes. embryonic or foetal tissue in addition to placental tissue visible.
- Dead foetuses: Hypoxemic foetuses which did not breathe spontaneously after the uterus had been opened.

Fetal examinations:

- External examinations: Yes: At necropsy each foetus was weighed and examined macroscopically for any external findings. Furthermore, the viability of the foetuses and the condition of the placentae, the umbilical cords, the foetal membranes and fluids were examined. Individual placental weights were recorded.
- Soft tissue examinations: Yes: After the foetuses had been sacrificed by CO2 , the abdomen and thorax were opened in order to be able to examine the organs in situ before they were removed. The heart and the kidneys were sectioned in order to assess the internal structure.
The sex of the foetuses was determined by internal examination of the gonads.
- Skeletal examinations: Yes: After the soft tissue examination all foetuses were placed in ethyl alcohol for staining of the skeletons (with the possible exception of the skulls according to a modified method of Dawson. The stained skeletons were placed on an illuminated plate and examined, evaluated and assessed. After the examination the stained skeletons were retained by litter.
- Head examinations: Yes: If heads of foetuses revealed severe findings (e.g. anophthalmia, microphthalmia, hydrocephalus, or cleft palate), the heads of these foetuses were severed from the trunk, fixed in Bouin's solution and later processed and assessed according to WILSON's method. About 10 transverse sections were prepared per head. After the examination the heads treated in this way were discarded.

- Evaluation criteria for assessing skeletons and organs of the foetuses:
There are differing opinions on the classification and assessment of changes in foetuses. Müntefering differentiates between malformations, which he defines as "severe morphological defects outside the range of variation of the species", and macroscopic/microscopic anomalies, which he defines as "slighter morphological deviations from normal". If these changes occur, they are regarded by Müntefering as a teratogenic effect.
However, transition from variation to malformation is fluid, and the term "slighter morphological deviation" is not defined. Neubert, on the other hand, tends to describe a change as a morphological abnormality, anomaly or functional anomaly instead of malformation since malformation refers primarily to gross-pathological changes.
In the present investigations the following terms (definitions) were used for describing a change:
- Malformations (concerning external, soft tissue and skeletal observations): Rare and/or probably lethal changes were classified as malformations (e.g. exencephaly, atresia ani, hernia umbilicalis).
- Variations (concerning external, soft tissue and skeletal observations): Changes which occur regularly also in control groups and have generally no adverse effect on survival were regarded as variations (e.g. dilated renal pelvis).
- Retardations (concerning skeletal observations only): Delays in skeletal development compared with the norm at the time of the examination were considered to be retardations (e.g. sternebra(e) not ossified).
- Unclassified observations (concerning external and soft tissue observations, only): External or soft-tissue observations, which could not be classified as malformations or variations (e.g. focal liver necrosis in foetuses).
According to the definitions specified before, the findings obtained in foetuses were classified accordingly.

Statistics:

The data were evaluated statistically using the computer systems of the Department of Toxicology of BASF Aktiengesellschaft.

Examinations of dams and foetuses:
Dunnett's Test was used for statistical evaluation of food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), weight of the uterus before it was opened, weight of foetuses, weight of placentae, corpora lutea, implantations, pre- and post-implantation losses, resorptions and live foetuses.
Fisher's Exact Test was used for statistical evaluation of conception rate, mortality (of the dams) and all foetal findings.

Indices:

Calculations of conception rate and pre- and post-implantation losses were carried out:
- The conception rate (in %) was calculated according to the following formula:

(number of pregnant animals / number of fertilised animals) x 100

The pre-implantation loss (in %) was calculated* according to the following formula:

((number of corpora lutea - number of implantations) / number of corpora lutea) x 100

The post-implantation loss (in %) was calculated* from the following formula:

((number of implantations - number of live foetuses)) / number of implantations) x 100

* Calculation on the basis of each individual pregnant animal with scheduled sacrifice

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

One doe (No. 21) of the low dose group (5 mg/kg bw/day) died spontaneously on day 7 p.i. immediately after the first gavaging (but without any clear indications for misgavaging). Furthermore, two does showed minor skin lesions in the laryngeal area (Nos. 46 and 56 (50 mg/kg bw/day)). There were no abnormal clinical findings for any other does in the study.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One low dose dam (No. 21) was found dead on day 7 p.i. Although this female died immediately after the first gavaging, no clear indications for misgavaging, but some unspecific lung lesions were found at necropsy.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no adverse effects on body weights or body weight changes which could be attributed to the oral administration of the test material. All values are within the range of biological variation.
The results of the corrected body weight gain (terminal body weight on day 29 p.i. minus weight of the uterus before it was opened minus body weight on day 7 p.i .) do not show any dose-response relationship and no differences of biological relevance between the groups.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption of the test material-treated dams was not influenced by the test material administration. All differences between the groups including the statistically significantly increased food consumption of the high dose group (50 mg/kg bw/day) on days 27 - 28 p.i. (post treatment period) and the statistically significantly reduced food intake of the 5 and 20 mg/kg does on days 0 - 7 p.i. are assessed as being of spontaneous nature and without any biological significance.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no substantial differences concerning the uterus weights between the controls and treatment groups (5, 20 and 50 mg/kg bw/day). All these values lie within the range of biological variation. This includes the relatively low mean gravid uterus weight in the 50 mg/kg group, which is caused by an incidentally lower number of live foetuses/doe in comparison to the control group.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At necropsy none of the does of the treatment groups (5, 20 or 50 mg/kg bw/day) showed any test material-induced finding.
Only some spontaneous necropsy findings were recorded for single animals of all dose groups including the controls. Most of these findings have to be related to the sacrifice of the animals (lungs with oedema and/or marginal emphysema); moreover, agenesia of gallbladder (animal No. 11 (control)), lung with focal haemorrhages in one lobe (animal No. 21 (5 mg/kg bw/day) which died intercurrently), skin lesions (dams Nos. 46 and 56 (50 mg/kg bw/day)) and blind ending uterine horns (dam No. 49 (50 mg/kg bw/day)) were observed.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

Only pregnant dams were used for the calculations of mean maternal food consumption, body weight and body weight change. Only pregnant dams with scheduled sacrifice (day 29 p.i .) were taken for the calculation of mean gravid uterine weights, mean net maternal body weight change (corrected body weight gain) and summary of reproduction data.
In this study, the following females were partly or totally excluded from the above mentioned calculations:
Control group (0 mg/kg bw/day): None
Low dose group (5 mg/kg bw/day): Female No. 70192 (21) - died intercurrently.
Intermediate dose group (20 mg/kg bw/day): None
High dose group (50 mg/kg bw/day): Female No. 70076 (49) - not pregnant.

Number of abortions:

not specified

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Concerning all groups, there were no test material-related and/or statistically significant differences in the values calculated in the mean number of corpora lutea, for the implantation sites or pre- and the post-implantation losses. The differences evident are considered to be incidental and within the normal range of deviations for animals of this strain and age.

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

Concerning all groups, there were no test material-related and/or statistically significant differences in the number of resorptions. The differences evident are considered to be incidental and within the normal range of deviations for animals of this strain and age.

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

There was a slight, statistically significant increase in the number of late resorptions at 50 mg/kg bw/day. However, the total number of late resorptions in this group (5 of a total of 96 implantations) was small, and in the context of the overall post-implantation loss, was considered not to be associated with treatment. The respective values calculated for the high dose group are fully in the historical control.

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

Concerning all groups, there were no test material-related and/or statistically significant differences in viable foetuses. The differences evident are considered to be incidental and within the normal range of deviations for animals of this strain and age.
The mean number of live foetuses/doe is lowest in the 50 mg/kg group (5.9 live foetuses/doe), but this value is also totally in the historical control range (6.0 [4.5 - 7.2] live foetuses/doe).

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The conception rate varied between 93 % (high dose group (50 mg/kg bw/day)) and 100 % (control, low and intermediate dose groups (0, 5 and 20 mg/kg bw/day)).
Concerning all groups, there were no test material-related and/or statistically significant differences in conception rate. The differences evident are considered to be incidental and within the normal range of deviations for animals of this strain and age.

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Remarks on result:

other: No maternal toxicity noted at highest dose tested.

Abnormalities:

effects observed, non-treatment-related

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean foetal weights were not influenced by the oral administration of the test material. All values are within the range of biological variation and do not show any dose-response relationship.

Reduction in number of live offspring:

not specified

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the foetuses in the treatment groups (5, 20 and 50 mg/kg bw/day) was comparable with the control foetuses. The differences observed in comparison to the control are without any biological relevance.

Changes in litter size and weights:

not specified

Changes in postnatal survival:

not specified

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No malformations were seen during the external examination of the foetuses. Only one type of external variation (pseudoankylosis) was found, which occurred without any relation to dosing. It was seen in single foetuses of all groups except the high dose group (50 mg/kg bw/day). There were no so-called unclassified observations (like placentae fused) in any of the foetuses.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Malformations of the foetal skeletons were noted for 2 out of 97 control foetuses (in 1 out of 14 litters) and 1 out of 96 intermediate dose foetuses (in 1 out of 15 litters). These malformations were related to the vertebral column (cervical/thoracic vertebral body/bodies fused and/or of irregular shape) and the ribs (fused).
The variations elicited were related to the skull (splitting of skull bones, epactal bone between nasal and frontal bones), the ribs (accessory 13th rib(s) and rudimentary cervical rib(s)), the vertebral column (accessory thoracic vertebra) and the sternum (sternebra(e) of irregular shape, fused or accessory sternebra). Most of these skeletal variations occurred without a clear dose response relationship and/or without any biologically relevant, statistically significant differences between the groups; however, the occurrence of fused or irregular- shaped sternebra(e) and consequently the foetal and litter incidence of overall skeletal variations was clearly increased in the 20 mg/kg group. Both findings were also found quite frequently in the historical control data. Therefore and because no dose-response relationship is present, the increased number of foetuses with skeletal variations in the intermediate dose group is assessed as being of spontaneous nature.
In all groups signs of retardations (incomplete or missing ossification of skull bones, vertebral column and sternebra(e)) were found; they occurred in a comparable frequency in the control and the test material-treated groups.
All differences between the groups concerning foetal skeletal retardations are without any biological relevance.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One control and one high dose foetus each showed soft tissue malformations.
For the control foetus agenesia of gallbladder was observed, while the high dose foetus had a septal defect, which led also to a dilated aortic arch and a dilated aorta descendens. All soft tissue malformations mentioned before are also present at a low incidence in the historical control data and are assessed as being of spontaneous nature.
Variations were detected in each group including the control, all occurred without a clear dose-response relationship and/or can be found at a comparable incidence in the historical control data. Aside from a separated origin of carotids, a very common finding in the rabbit strain used, another soft tissue variation (heart with traces of interventricular foramen/septum membranaceum) was also found quite frequently.
Furthermore, one foetus of the low and intermediate dose group each (5 and 20 mg/kg bw/day) exhibited hypoplasia of gallbladder and another one of test group 2 (20 mg/kg bw/day) showed dilated renal pelvis.
One control foetus showed a so-called unclassified observation (focal liver necrosis).

Other effects:

no effects observed

Description (incidence and severity):

The mean placental weights in the treatment groups (5, 20 and 50 mg/kg bw/day) were not influenced by the test material administration. The differences observed in comparison to the control are without biological relevance and lie within the range of biological variation.

Details on embryotoxic / teratogenic effects:

With the exception of skeletal variations there are no statistically significant differences between the control and the test material-treated groups with respect to external, soft tissue, skeletal or total malformations, variations and/or retardations. Almost all findings are without a clear dose-response relationship and/or appear to about the same extent either in the actual or in the historical control data. The statistically significantly increased occurrence of fused or irregular shaped sternebra(e) in the foetuses of the 20 mg/kg group is without any biological relevance and is assessed as being of spontaneous nature.

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: No teratogenic effects noted at highest dose tested

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

Under the conditions of the study the no observed adverse effect level (NOAEL) on the maternal and foetal rabbits is 50 mg/kg bw/day.

Executive summary:

The test material was tested for its prenatal toxicity in Himalayan rabbits according to OECD Test Guideline 414 and in compliance with GLP.

In a preceding range-finding study, the test material was administered as an aqueous suspension to groups of five nulliparous time-mated female Himalayan rabbits by stomach tube at doses of 0, 40, 80 and 120 mg/kg bw/day from day 6 to day 19 post insemination (p.i.). The dams were sacrificed on the day following the last oral test material administration (day 20 p. i.).

Treatment at 120 mg/kg bw/day was associated with death in 2 of 5 females. Treatment at 120 and at 80 mg/kg bw/day was associated with clear body weight loss during the treatment period, and clearly reduced food consumption. Treatment at 40 mg/kg bw/day was also associated with body weight loss and marginally lower food consumption.

Previous experience with this class of compound indicates that the dose response curve shows a rapid increase in toxic response over a very small range. Therefore a high dose of 50 mg/kg bw/day was selected for the present full-scale study at a dose level at which clear maternal toxic effects were expected.

In the full-scale study, the test material was administered as an aqueous suspension to 14 - 15 pregnant female rabbits/group by stomach tube at doses of 5, 20 and 50 mg/kg bw on day 7 through day 19 p.i.

A standard dose volume of 10 mL/kg body weight was used.

The control group was dosed with the vehicle only (0.5 % aqueous carboxymethyl cellulose solution).

Food consumption and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day.

On day 29 post insemination, all surviving females were sacrificed and assessed by gross pathology. The foetuses were dissected from the uterus, sexed, weighed and further investigated for any external, soft tissue and skeletal findings.

There were no substantial material-related effects on the does concerning food consumption, body weight, body weight change, uterine weights, corrected body weight change, clinical and necropsy observations up to and no differences of biological relevance between the control and the material-treated groups (5, 20 and 50 mg/kg bw/day) in conception rate, mean number of corpora lutea, total implantations, resorptions and live foetuses, foetal sex ratio as well as in the values calculated for the pre- and the post-implantation losses. No dose- and/or material-related differences were recorded for placental and foetal body weights.

The external, soft tissue and skeletal examination of the foetuses revealed no differences between the control and the test material-treated groups which might be related to the test material administration. Number and type of the foetal external, soft tissue and skeletal findings, which were classified as malformations, variations and/or retardations, recorded for the 5, 20 and 50 mg/kg foetuses were substantially similar to actual and/or historical control values.

No signs of embryo-/foetotoxicity were noted in the present full-scale study, especially no indications of any teratogenicity occurring up to and including the highest dose level.

Under the conditions of the study the no observed adverse effect level (NOAEL) on the maternal and foetal rabbits is 50 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Two studies both awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997) were conducted according to OECD Test Guidlines and in compliance with GLP.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Developmental Toxicity in Rats: Hellwig (1993)

The test material was assessed for its prenatal toxicity in Wistar rats according to OECD Test Guideline 414 and in compliance with GLP. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was administered as an aqueous suspension to 20 - 24 pregnant female rats/group by stomach tube at doses of 20, 50 and 100 mg/kg bw on day 6 through day 15 post coitum (p.c.). A standard dose volume of 10 mL/kg body weight was used. The control group was dosed with the vehicle only (0.5 % aqueous carboxymethyl cellulose solution).

Food consumption and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day.

On day 20 post coitum, all females were sacrificed and assessed by gross pathology. The foetuses were dissected from the uterus, sexed, weighed and further investigated for any external, soft tissue and/or skeletal findings. The following findings were obtained and assessed as test material-related:

High dose dams (100 mg/kg bw/day):

- Statistically significantly reduced food consumption at the beginning of treatment period (days 6 - 10 p.c.);

- Statistically significantly lower mean body weights than the controls on day 8 p.c.; slight body weight loss on days 6 - 8 p.c. and, if calculated for the whole treatment period, a weight gain of about 18 % less than that of controls;

- Reduced corrected body weight gain;

- Statistically significantly increased number of foetuses with a skeletal variation (rudimentary cervical rib(s)).

Intermediate dose dams (50 mg/kg bw/day):

- No test material-related effects on dams or foetuses.

Low dose dams (20 mg/kg bw/day):

- No test material-related effects on dams or foetuses.

Thus, under the conditions of this full-scale study, the test material caused some overt signs of maternal toxicity and marginal signs of embryo-/foetotoxicity at 100 mg/kg bw/day. No test material-induced teratogenic effects were observed up to and including the dose of 100 mg/kg bw/day. There were no test material-induced, adverse effects on dams or foetuses of the intermediate or low dose groups (50 or 20 mg/kg bw/day).

In the preceding range-finding study, however, which was carried out under comparable study conditions (e.g. same batch, same rat strain, same treatment schedule), massive signs of maternal toxicity in the form of clearly reduced food consumption, retarded body weight gain, impairment of several haematological and clinicochemical parameters and/or increased liver and kidney weights were found at doses of 100 and 150 mg/kg bw/day, whereas marginal maternally toxic effects were still present at 50 mg/kg bw/day.

Under the conditions of the study the no observed adverse effect level (NOAEL) on maternal and foetal rats is 50 mg/kg bw/day.

Developmental Toxicity in Rabbits: Hellwig (1993)

The test material was tested for its prenatal toxicity in Himalayan rabbits according to OECD Test Guideline 414 and in compliance with GLP. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

In a preceding range-finding study, the test material was administered as an aqueous suspension to groups of five nulliparous time-mated female Himalayan rabbits by stomach tube at doses of 0, 40, 80 and 120 mg/kg bw/day from day 6 to day 19 post insemination (p.i.). The dams were sacrificed on the day following the last oral test material administration (day 20 p. i.).

Treatment at 120 mg/kg bw/day was associated with death in 2 of 5 females. Treatment at 120 and at 80 mg/kg bw/day was associated with clear body weight loss during the treatment period, and clearly reduced food consumption. Treatment at 40 mg/kg bw/day was also associated with body weight loss and marginally lower food consumption.

Previous experience with this class of compound indicates that the dose response curve shows a rapid increase in toxic response over a very small range. Therefore a high dose of 50 mg/kg bw/day was selected for the present full-scale study at a dose level at which clear maternal toxic effects were expected.

In the full-scale study, the test material was administered as an aqueous suspension to 14 - 15 pregnant female rabbits/group by stomach tube at doses of 5, 20 and 50 mg/kg bw on day 7 through day 19 p.i.

A standard dose volume of 10 mL/kg body weight was used.

The control group was dosed with the vehicle only (0.5 % aqueous carboxymethyl cellulose solution).

Food consumption and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day.

On day 29 post insemination, all surviving females were sacrificed and assessed by gross pathology. The foetuses were dissected from the uterus, sexed, weighed and further investigated for any external, soft tissue and skeletal findings.

There were no substantial material-related effects on the does concerning food consumption, body weight, body weight change, uterine weights, corrected body weight change, clinical and necropsy observations up to and no differences of biological relevance between the control and the material-treated groups (5, 20 and 50 mg/kg bw/day) in conception rate, mean number of corpora lutea, total implantations, resorptions and live foetuses, foetal sex ratio as well as in the values calculated for the pre- and the post-implantation losses. No dose- and/or material-related differences were recorded for placental and foetal body weights.

The external, soft tissue and skeletal examination of the foetuses revealed no differences between the control and the test material-treated groups which might be related to the test material administration. Number and type of the foetal external, soft tissue and skeletal findings, which were classified as malformations, variations and/or retardations, recorded for the 5, 20 and 50 mg/kg foetuses were substantially similar to actual and/or historical control values.

No signs of embryo-/foetotoxicity were noted in the present full-scale study, especially no indications of any teratogenicity occurring up to and including the highest dose level.

Under the conditions of the study the no observed adverse effect level (NOAEL) on the maternal and foetal rabbits is 50 mg/kg bw/day.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to reproductive or developmental toxicity.