Prometryn

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

- Oral, 2-generation study (EPA 83-4, OECD TG 418), Giknis 1990
lowest, average NOAEL for parental, systemic toxicity 0.68 mg/kg bw/day in P1 males;
lowest, average NOAEL for reproduction toxicity 99.6 mg/kg bw/day in P1 males and 105.0 mg/kg bw/day in P1 females;
lowest, average NOAEL for foetal toxicity 53.1 mg/kg bw/day in F1 males and 53.6 mg/kg bw/day in F1 females

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 Nov 1987 to 12 Sep 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

Version / remarks:

November 1982

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 418

Version / remarks:

May 12, 1981

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl: CD BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) males 38 wks and females 37 wks; (F1) 14 wks after weaning
- Weight at study initiation: (P) Males: 171-256 g; Females: 141-201 g; (F1) Males: 139-215 g; Females: 127-177 g
- Housing: rats were caged indivivually (except during the mating, lactation, and weaning periods) in solid bottom cages containing hardwoord chips which were changed weekly
- Diet: Certified Rodent Chow (or its admixture) ad libitum
- Water: tap water ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26
- Humidity (%): 30-70

IN-LIFE DATES:
- P0: From: 4 Nov 1987 To: 29 March 1988
- P1: From: 10 Feb 1988 To: 12 Sep 1988

Route of administration:

oral: feed

Vehicle:

acetone

Remarks:

evaporated overnights

Details on exposure:

DIET PREPARATION
Homogeneous blends of the test material in the diet were prepared. The substance was disscoved in acetone and premixed with an appropriate amount of feed. The acetone was evaporatied overnights and the resulting premix was used to prepare the test substance/feed admixturered. The control feed admixture was prepared in a similar manner.

Details on mating procedure:

P0 animals each groups were housed together in a 1:1 (male to female) ratio for mating. Animals were allowed 3 weeks in which to mate and were separated on the day that evidence of mating (sperm and/or copulatory plug) was observed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability studies on admixtures of the test substance in rodent chow over the concentration range of 10 ppm to 50,000 ppm indicated that these mixtures were chemically stable for at least 51 days at room temperature. Analyses of multiple subsamples of test admixtures established acceptable homogeneity. The test substance ranged from 93.2% to 106.4% for these homogeneity analyses. The results of the periodic concentration analyses, performed prior to dosing for admixtures prepared for weeks 1, 5, 9, 11, 17, 18, 25, 33 and 41, showed the concentrations of the test substance in the admixtures to be 93% to 108% of the expected values based on the average results obtained from two or more analyses.

Duration of treatment / exposure:

7 days per week for 2 parental generations of animals and their offspring throughout all phases of the study

Frequency of treatment:

Continuously

Details on study schedule:

The first day of P0 exposure was designated day "0". Following almost 11 weeks of exposure to the appropriate diet. P0 animals each groups were housed together in a 1:1 (male to female) ratio for mating. Animals were allowed 3 weeks in which to mate and were separated on the day that evidence of mating (sperm and/or copulatory plug) was observed. P0 females were allowed to deliver litters.
Following the weaning of the last litter, 30 male and 30 female F1 pups were selected from the available pups in each group to become the second parental generation (P1). Wherever possible, one pup of each sex was randomly selected from each available litter. Litters from which additional pups were selected (in order to achieve 30 per sex per group) were chosen at random. The initiation of the P1 premating phase began with the first recorded body weight. The remaining F1 pups were sacrificed and subjected to a gross internal examination.
One half of the P0 males were sacrificed on day 141 of study and the remaining half were sacrificed on day 142 of study. P0 females were sacrificed after weaning of the F1 pups, half on day 144 of study and half on day 145 of study.
Following their selection, the F1 (P1) animals were allowed 14 weeks of dietary exposure prior to mating. Animals were housed together in the same manner as P0 animals and allowed to deliver their litter. All F2 litters were sacrificed on lactation day 21 and 40 F2 (5/sex/group) were submitted to the Pathology subdivision for necropsy and tissue preservation.
One half of the F1 (P1) males were sacrificed after 145 days on study and the second-half after 146 days on study. One-half of F1 (P1) females were sacrificed after 169 days and the remaining one-half were sacrificed after 172 days.

Dose / conc.:

10 ppm

Remarks:

Group 2: 0.48-0.93, 0.45-1.09, 0.57-0.88 and 0.54-1.00 mg/kg bw day actual test substance consumption for P0 males, P1 males, P0 females and P1 females, respectively.

Dose / conc.:

750 ppm

Remarks:

Group 3: 36.04-70.19, 33.54-80.26, 42.48-64.68 and 43.66-76.04 mg/kg bw day actual test substance consumption for P0 males, P1 males, P0 females and P1 females, respectively.

Dose / conc.:

1 500 ppm

Remarks:

Group 4: 72.36-126.76, 68.78-168.88, 87.13-122.78 and 85.87-158.54 mg/kg bw day actual test substance consumption for P0 males, P1 males, P0 females and P1 females, respectively.

No. of animals per sex per dose:

30

Control animals:

yes, plain diet

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
All animals were observed daily for changes in appearance and behaviour and twice daily for mortality.

BODY WEIGHT
Body weight data was recorded weekly for the males during the premating period and was recorded weekly thereafter and at terminal sacrifice. Body weight data for the females was recorded weekly during the premating period and on days 0, 7, 14 and 20 of gestation. Female body weights during lactation were recorded on days 0, 7, 14 and 21 for the F0 (P0) generation. During lactation, the F1 (P1) generation female body weights were recorded on days 0, 4, 8, 14 and 21.
P0 females that showed positive evidence of mating but failed to deliver a litter were weighed during the period of presumed gestation and at terminal sacrifice.
F1 females that showed positive evidence of mating but failed to deliver a litter were weighed during the period of presumed gestation until terminal sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Feed consumption data was recorded weekly for the males during the premating period. Feed consumption for the females was recorded weekly during the premating period and on days 0, 7, 14 and 20 of gestation.
- Test article consumption was calculated as: Test article consumption = mean daily food consumption / average mean body for interval x dietary concentration

Litter observations:

POSTNATAL OBSERVATIONS OF THE F1 AND F2 GENERATIONS
On the day of delivery, the number and sex of viable pups and the number of stillborn pups were recorded. Newborns were examined for gross abnormalities.
On postnatal day 4, the litters were randomly culled to eight pups (four males and four females), whenever possible.
Pups were observed periodically for changes in appearance and behaviour, and deaths were recorded throughout the lactation period.

IN-LIFE DATA COLLECTION
In- life data for feed consumption, body weight, clinical signs, pup observations, pup sexes and pup weights were collected.
Pup body weights were recorded on lactation days 0, 4, 7, 12, and 21 for the F1 generation and on lactation days 0, 4, 8, 14 and 21 for the F2 generation

Postmortem examinations (parental animals):

All parental animals were sacrificed and necropsied by the Pathology Subdivision. Uterine implantation sites were counted and recorded at necropsy. Any grossly abnormal organ(s) and specimens of vagina, cervix, uterus, ovaries, testes, epididymides, seminal vesicles, prostate, pituitary and coagulating gland were collected from parental animals and fixed in 10% neutral buffered formalin. The fixed tissues from the control and high-dose animals were examined microscopically. These same tissues were examined from 40 randomly selected F2 pups. Grossly abnormal tissues were also examined microscopically. Paired testes and ovaries were weighed.

Postmortem examinations (offspring):

All pups removed from each litter at the time of culling were sacrificed and subjected to a gross internal inspection. This inspection included a brief examination of the abdominal and thoracic cavities, confirmation of sex and a slice through each kidney.
The Pathology Subdivision performed necropsies on 40 (5/sex/group) randomly selected F2 pups.

Statistics:

Statistical analyses were performed as indicated below unless otherwise stated:
- Parental Body Weight, Body Weight Gain, Feed Consumption, Absolute and Relative Organ Weight: One-way Analysis of Variance (ANOVA, Snedecor and Cochran, 1968), Bartlett's Test for Homogeneity of Variance (Snedecor and Cochran, 1968)and Dunnett's Method of Multiple Comparisons between control and treatment groups (Dunnett, 1955, 1964). The calculations derived from the ANOVA were employed in the subsequent Bartlett's and Dunnett's Tests. The primary focus of these analyses is on the results of the Dunnett's comparisons between the control and each of the treated groups. Statistically significant differences between treatment groups (e.g. low dose vs. high-dose group) are not discussed.
- Precoital Interval, Maternal Gestation Duration, Number of Implantations, Viable Fetuses, Stillbirths; Calculated Post- implantation Loss, % Post-implantation Loss, % Stillbirths, Survival Indices, Fertility Indices and Pup Sex Ratios (% males): Chi-squared distributed statistics or Mantel’s trend test
- F1 and F2 generation pup body weights: Healy Analysis (Healy, 1972)

Reproductive indices:

- Post-implantation loss = No. implantations – No. viable pups
- % Post-implantation loss = (No. implantation sites – No. viable pups) / No. implantation sites per dam x 100
- Female fertility = No. pregnant females / No. females with positive evidence for mating x 100
- Female mating index = No. females with positive evidence of mating / No. females cohoused x 100
- Gestation index = No. live litters / No. pregnant females x 100
- Male fertility = No. fertile males / No. males with positive evidence of mating x 100
- Male mating index = No. males with positive evidence of mating / No. males cohoused x 100

Offspring viability indices:

- pre-culling survival = No. pups surviving to day 4 (pre-cull) lactation / No. pups alive day 0 lactation x 100
- post-culling survival = No. pups alive at day 21 lactation / No. pups alive day 4 (post-cull) lactation x 100
- percent males = Total No. viable males (day 0 lactation per group) / Total No. viable pups (day 0 lactation per group) x 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no compound-related clinical observations in this generation, only incidental clinical observations.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no compound-related mortalities in this generation. There were three unscheduled deaths in the P0 generation. One control female (group 1) with a tissue mass (mammary gland adenocarcinoma) was sacrificed in poor physical condition on study day 75 and two 750 ppm (group 3) females were found dead on day 99 and day 102, respectively. The death of one female of group 3, was not associated with any clinical or necropsy observations, while the other female of group 3 was noted as having a dark thick, yellow exudate from its bladder and at necropsy was found to have a bilateral infarction of the kidneys. These deaths were not considered to be treatment-related based on the low incidence of occurrence and a review of the clinical, necropsy, and pathology findings.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Males: Mean P0 male body weights in the 10 ppm and 1500 ppm groups were comparable to controls on premating day 0; however a slight, yet statistically significant baseline difference of approximately 3% was observed in the 750 ppm group. This baseline difference persisted throughout the premating period and resulted in statistically lower mean body weights in the 750 ppm group on premating days 7, 21, 28, 35 and 56. These lower mean body weights were generally not associated with reductions in body weight gain or feed consumption and were therefore not related to compound. Compound-related, statistically significant reductions in mean body weight were observed in the 1500 ppm group males on premating days 7 and on all assessment occasion during days 21-70; while significant reductions in mean body weight gain were noted for premating days 0-7, 14-21, 28-35, 49-56 63-70, and overall mean premating body weight gain (premating days 0-70). Non-treatment related, yet statistically significant, fluctuations in mean body weight gain were noted in the 750 ppm group males on premating days 14-21 and 63-70. Group mean body weights were significantly reduced in the 1500 ppm group males during mating and postmating on days 77, 84, 91, 98, 105, 112, 119, 126 and 133; and at terminal necropsy (days 141 or 142). These reductions ranged from 6.3% to 8.2% and were attributed to treatment with the test substance. Although slight, group mean body weight in the 750 ppm group males was also significantly reduced on days 77 and 84. The lower mean weights detected in the 750 ppm group averaged approximately 4% less than controls and were most likely the result of the initial lower mean body weights observed in this group and not a compound effect. The 1500 ppm group mean overall body weight gain (from day 0- termination; was significantly reduced by approximately 12% and mean terminal body weight was significantly reduced by approximately 7.5%. These reductions were attributed to treatment with the test substance No apparent effect on overall mean bodyweight gain or mean terminal body weight was observed at 10 or 750 ppm.
- Females (pre-mating): The 1500 ppm group mean female pre-mating body weights tended to be lower than controls after the first week of premating (2-7%) with a statistically lower mean body weight observed on day 70. In addition the 1500 ppm group mean body weight gain tended to be lower than controls with statistically reduced mean body weight gains detected during premating days 0-7, 42-49 and for mean overall premating weight gain, days 0-70. These reductions in mean body weight parameters occurred concomitantly with treatment-delated reductions in food consumption and were also attributed to treatment. The 10 ppm and 750 ppm group mean body weight and body weight gain were unaffected by treatment. A spurious, yet statistically significant reduction in mean body weight gain was observed at 10 ppm during premating days 63-70. As this decrease was not associated with decreased food consumption or body weight. It was not considered to be toxicologically significant.
- Females (gestation): The 1500 ppm females had consistently lower mean body weights during gestation with treatment-related, statistical decreases observed on gestational days 0, 7, 14 and 20. During gestation mean body weight gain tended to be less than controls at 1500 ppm on days 0-7, 14-20 and 0-20; whereas it was unaffected at ≤ 750 ppm as mean body weight. A spurious, yet statistically significant increase in mean overall gestational body weight gain (days 0-20) was observed at 10 ppm and was not considered to be toxicologically significant.
- Females (lactation): The 1500 ppm animals had significantly reduced mean body weight on lactation day 0 and 14, while significant increases in mean weight gain were observed during lactation days 0-7, 14-21, and 0-21.
- Females (TBW): There were no statistically significant effects on mean terminal body weight in P0 females in this study; although the 1500 ppm group mean terminal body weight was less than the control value by approximately 5%.

See Table 4, 5, 6, 7 and 8 in “Any other information on results incl. tables” for more information.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Males: Slight, but statistically significant, treatment-related reductions in mean food consumption of approximately 5-13% were observed in the 1500 ppm group males as compared to controls at the following intervals during the premating period; days 0-7, 21-28, 35-42, 42-49, 49-56 and 63- 70. An isolated, slight, reduction of approximately 5% was observed in the 750 ppm group males during premating days 0-7 and was statistically significant. This reduction was most likely related to initial palatability problems of the dietary admixture upon initial exposure and was not of toxicological significance. No effect on mean food consumption was observed in the 10 ppm group males. The mean of the daily doses of test article ingestion on a mg/kg basis were approximately proportional to dietary concentration increments.
- Females: A statistically significant compound-related reduction in mean food consumption of approximately 9% was observed at 1500 ppm during gestation days 0-7. Mean food consumption was comparable among all groups for the remainder of gestation. No effect on mean food consumption was noted in either the 10 ppm or the 750 ppm group females at any time during the study. The means of daily doses on a mg/kg basis were approximately proportional to dietary concentration increments.

See Table 1, 2 and 3 in “Any other information on results incl. tables” for more information.

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

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related histopathological findings in P0 animals.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no significant treatment-related effects on mating as the precoital interval was similar among all groups. The duration of gestation, the reproductive parameters and the mating, fertility, and gestation indices were comparable among the control and substance-treated groups.

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

750 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: dietary equivalent to 36.04 - 70.19 mg/kg bw/day for males and 42.48 - 64.68 mg/kg bw for females.

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

>= 1 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: dietary equivalent to 72.36 - 126.76 mg/kg bw for males and 87.13 - 122.78 mg/kg bw/day for females

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no compound-related clinical observations.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no compound-related deaths. Three females did not survive the postmating phase of the study. Two females, one from the 10 ppm group and one from the 750 ppm group were observed in dystocia prior to sacrifice on gestation days 25 and 26 respectively, and another 10 ppm group female was lean cold to touch had a red nasal discharge, paralyzed hindlimbs and a bloody swollen vagina prior to sacrifice on study day 143 and was found to have pyelonephritis of one kidney on histological examination. The first two sacrificed females were attributed to non-treatment related difficulties in labour and delivery. The last sacrificed female was to avoid unnecessary discomfort to the animal caused by non- treatment- related pyelonephritis.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Males: Treatment-related reductions in mean body weight were observed in the 750 ppm groups throughout the premating, mating and postmating phases of the study with statistical differences recorded for all assessment occasions during days 28-140 in the 750 ppm group and during days 0- 140 at 1500 ppm and at termination (days 145 or 146) in both groups. Reductions in mean body weight were accompanied by reduced mean body weight gain in both the 750 and 1500 ppm groups during the premating period. These reductions were statistically significant for all assessment intervals during days 0-7, 14-21, 28-35, 0-98 and 0-terminal body weight in the 750 ppm group and during days 0-21, 28-42, 70-77, 0-98, and 0-terminal body weight in the 1500 ppm group. Body weight parameters were unaffected in the 10 ppm group.
- Females: Body weights in the 750 ppm and 1500 ppm groups tended to be lower than controls throughout most of the study. Significant treatment-related reductions in mean body weight were observed in the 750 ppm group during premating (days 49-98)and at terminal necropsy and in the 1500 ppm group during premating (days 21-98) gestation (days 0-20), and lactation (day 0) periods as well as at terminal necropsy. Mean body weight gains were reduced in the 750 ppm and 1500 ppm groups females during the majority of the premating period and were significantly reduced at 750 ppm during premating days 42-49 and overall days 0-98 and at 1500 ppm during premating days 21-35 and 42-49 and overall days 0-98. During gestation body weight gains were comparable among all groups. Lactation body weight gains tended to be greater at 750 ppm and 1500 ppm with mean lactation body weight gain significantly greater during days 0-8, 14-21, as well as mean overall lactation weight gain (days 0- 21) at 1500 ppm; while mean overall lactation body weight gain (day 0-21) was significantly greater at 750 ppm when compared to controls. Mean body weight parameters were unaffected throughout the study at 10 ppm. A single incidental increase in mean body weight gain was observed at 10 ppm during premating days 63-70.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Males: Statistically significant treatment-related reductions in mean food consumption were observed on all assessment occasions from premating days 7-14 to 91-98 in the 750 ppm group (6-13%) and premating days 0-7 to 91-98 at 1500 ppm (10-16%). Food consumption at 10 ppm was unaffected by the test substance. The means of daily doses on a mg/kg basis were approximately proportion- al to dietary concentration increments.
- Females (premating): As a result of treatment, mean food consumption in the 750 ppm and 1500 ppm groups tended to be lower than controls throughout the premating period. Statistically significant reductions of 9-13% were observed at 750 ppm over premating days 28-63 and 91-98, while significant reductions of 9-16% were observed at 1500 ppm over premating days 14-21, 28-77 and 84-98. The 10 ppm group female food consumption was unaffected by treatment; however a single, spurious reduction in food consumption (8%) was observed at 10 ppm during days 91-98. In the absence of any noteworthy body weight effects or clinical observations this finding was not considered to be biologically significant. The means of the daily doses on a mg/kg basis were approximately proportional to dietary concentration increments.
- Females (gestation): Mean food consumption during gestation approximated control values on all assessment occasions in the 10 ppm and 750 ppm groups. In the 1500 ppm group mean food consumption was 7-9% less than controls with statistical significance detected for days 0-7. The lower food consumption values at 1500 ppm were attributed to treatment. The means of the daily doses on a mg/kg basis were approximately proportional to dietary concentration increments

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 treatment-related effects on mean absolute ovary or testes weight or relative mean ovary weight in the P1 generation; however, in the 1500 ppm group relative mean testes weights were increased as a consequence of reduced mean terminal body weight noted at this dose level. Additionally, the mean absolute testes weight was decreased at 750 ppm but this decrease was attributed to the smaller size of the males in this group and not treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related necropsy observations in the P1 generation parental animals.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related histologic findings in the P1 animals.

Histopathological findings: neoplastic:

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):

There were no treatment-related effects on mating as the precoital interval was similar for all groups. Male and female mating and fertility indices, the duration of gestation and the gestation index were comparable among the control and treated groups. There were no treatment-related effects on any reproductive parameters examined. However, the mean number of implantation sites and the mean number of viable pups were significantly increased in the 750 ppm and 1500 ppm groups. These increases were incidental to treatment. All other reproductive parameters including the number of stillborn pups, the percentage of stillborn pups, calculated post-implantation loss and percentage post-implantation loss were comparable among all groups.

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

10 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: dietary equivalent to 0.45 - 1.09 mg/kg bw/day for males and 0.54 - 1.00 mg/kg bw/day for females

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

>= 1 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: dietary equivalent to 68.78 - 168.88 mg/kg bw/day for males and 85.87 - 158.54 for females

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no F1 pup clinical observations in this study.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no treatment related effects on F1 litter size.
Survival indices were comparable among the control and substance-treated groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Pup weights were reduced at 1500 ppm for both sexes on all assessment occasions lactation days 0, 4 (pre-cull, post-cull) 7, 14, and 21. Pup weights at 750 ppm also tended to be less than controls during lactation and were significantly reduced on lactation days 14 and 21. The lower pup weights observed through day 7 of lactation in the 750 ppm group were thought to be due to a slightly larger litter size prior to culling. In the 1500 ppm group the lower pup weights were also influenced by a slightly larger litter size and may have been compounded by maternal toxicity. Toward the end of lactation, the lower pup weights were considered due to ingestion of the substance feed admixtures by both the 750 ppm and 1500 ppm pups.

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:

no effects observed

Description (incidence and severity):

There were no treatment related effects on pup sex ratios.

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:

no effects observed

Description (incidence and severity):

There were no compound- related necropsy findings. Following the selection of the P1 generation, the F1 pups not selected for the second parental generation were sacrificed by subjected to a gross internal inspection. All findings were incidental.

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

750 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: 33.54 - 80.26 mg/kg bw

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no compound-related clinical signs in pups of the F2 generation.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no treatment-related effects on either parameter; however as a consequence of the incidental increase in the mean number of live foetuses in the 750 ppm and 1500 ppm groups, mean litter size on day 0 was also greater in both groups than the controls. This finding was considered to be incidental.
Survival indices were comparable among the treated and the control groups at both intervals assessed, days 0-4 (pre-cull) or days 4-21 (post-cull) regardless of whether the sexes were analysed separately or as pooled values.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related reductions in mean body weight were observed in the 1500 ppm group F2 male and female pups during the majority of the post-natal period [days 4 (pre- and post-cull) 14 and 21]. These lower pup weights were influenced by both a slightly larger litter size and maternal toxicity. The 10 ppm and 750 ppm group F2 pup weights were similar to controls.

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:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on either parameter.

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:

no effects observed

Description (incidence and severity):

No compound-related necropsy findings were observed. No treatment-related gross alterations were observed in the F2 pups.

Histopathological findings:

no effects observed

Description (incidence and severity):

No treatment-related microscopic alterations were observed in the F2 pups.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

750 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: 33.54 - 80.26 mg/kg bw

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

1 500 ppm

Treatment related:

yes

Relation to other toxic effects:

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

Dose response relationship:

yes

Relevant for humans:

yes

Table 1. Summary of P0 Male Pre-Mating Food Consumption (grams/day) (Mean ± SD)

Interval	Study days	Dietary level ppm
		Control (0)	10	750	1500
1	0-7	23.65 ± 2.19 (30)$	22.75 ± 1.96 (30)	22.39 ± 1.33* (30)	20.48 ± 1.80* (30)
2	7-14	23.92 ± 1.78 (30)	23.81 ± 1.78 (30)	23.28 ± 1.61 (30)	23.26 ± 2.22 (30)
3	14-21	24.12 ± 2.09 (30)	24.25 ± 1.84 (29)	23.79 ± 1.58 (30)	23.54 ± 2.05 (30)
4	21-28	25.02 ± 2.21 (30)	25.26 ± 1.96 (28)	24.55 ± 1.57 (29)	23.70 ± 2.17* (30)
5	28-35	24.58 ± 2.12 (28)	25.07 ± 2.13  (30)	24.02 ± 1.69 (30)	23.40 + 2.07 (30)
6	35-42	25.44 ± 2.13 (30)	25.91 ± 1.96 (30)	24.49 ± 1.92 (30)	24.01 ± 2.15* (30)
7	42-49	25.56 ± 2.42 (30)	25.66 ± 2.03 (30)	24.74 ± 2.08 (30)	24.07 ± 2.33* (30)
8	49-56	25.84 ± 2.76 (30)	25.69 ± 2.20 (30)	24.56 ± 1.79 (30)	23.79 + 2.46* (30)
9	56-63	25.49 ± 2.73 (30)	25.68 ± 2.33 (30)	24.08 ± 1.94 (30)	24.06 ± 2.26 (30)
10	63-70	26.01 ± 2.28 (29)	25.81 ± 2.55 (30)	24.74 ± 1.83 (30)	24.26 ± 2.46* (30)

$ Numbers in parenthesis ( ) equal number of animals used in mean

* Significantly different from Controls at P ≤ 0.05

 

Table 2. Summary of P0 Female Pre-Mating Food Consumption(grams/day) (Mean ± SD)

Interval	Study days	Dietary level ppm
		Control (0)	10	750	1500
1	0-7	16.05 ± 1.79 (30)$	15.86 ± 1.53 (30)	15.41 ± 1.27 (30)	13.87 ± 1.16* (30)
2	7-14	17.06 ± 2.07 (30)	17.11 ± 2.44 (30)	16.98 ± 1.38 (30)	15.98 ± 1.23 (30)
3	14-21	16.89 ± 2.15 (30)	16.72 ± 1.97 (30)	17.06 ± 1.49 (30)	16.16 ± 2.74 (28)
4	21-28	17.32 ± 2.16 (30)	17.35 ± 2.13 (30)	17.51 ± 1.84 (30)	16.23 ± 1.42 (30)
5	28-35	16.91 ± 2.10 (30)	17.30 ± 2.13 (30)	17.41 ± 1.84 (29)	15.51 ± 1.05* (29)
6	35-42	17.19 ± 1.76 (30)	17.14 ± 2.36 (30)	17.63 ± 2.16 (30)	15.97 ± 1.35* (30)
7	42-49	17.42 ± 2.38  (30)	17.96 ± 2.60 (30)	17.87 ± 2.19 (30)	15.97 ± 2.12* (30)
8	49-56	17.94 ± 2.56 (30)	17.26 ± 2.18 (30)	17.07 ± 2.49 (30)	15.59 ± 1.12* (30)
9	56-63	17.55 ± 2.50 (30)	16.86 ± 2.26 (30)	16.59 ± 1.70 (30)	15.40 ± 1.22* (30)
10	63-70	17.61 ± 2.32 (30)	16.93 ± 1.96 (30)	17.05 ± 1.94 (30)	15.89 ± 1.13* (30)

$ Numbers in parenthesis ( ) equal number of animals used in mean

* Significantly different from Controls at P ≤ 0.05

Table 3. Summary of P0 Female Gestation Food Consumption(grams/day) (Mean ± SD)

Interval	Days of Gestation	Dietary level ppm
		Control (0)	10	750	1500
1	0-7	22.22 ± 2.23 (29)$	21.19 ± 1.96 (24)	20.62 ± 2.21 (29)	19.23 ± 2.21* (29)
2	7-14	21.61 ± 2.80 (29)	21.55 ± 2.87 (24)	21.86 ± 1.92 (29)	20.77 ± 1.41 (29)
3	14-20	22.87 ± 2.83 (29)	23.42 ± 1.97 (24)	22.45 ± 3.49 (28)	21.53 ± 3.20 (29)

$ Numbers in parenthesis ( ) equal number of animals used in mean

* Significantly different from Controls at P ≤ 0.05

 

Table 4. Summary of P0 Male Pre-Mating Body Weights (grams) (Mean ± SD)

Study days	Dietary level ppm
	Control (0)	10	750	1500
0	220.67 ± 11.27 (30)$	218.57 ± 9.03 (30)	213.80 ± 12.71* (30)	221.27 ± 10.94 (30)
7	273.67 ± 15.10 (30)	271.03 ± 13.89 (30)	265.10 ± 10.96* (30)	262.83 ± 13.38* (30)
14	319.13 ± 16.81 (30)	320.70 ± 14.78 (30)	311.37 ± 12.60 (30)	310.03 ± 19.30 (30)
21	364.47 ± 20.62 (30)	364.93 ± 19.08 (30)	351.80 ± 13.74* (30)	348.23 ± 22.23* (30)
28	399.57 ± 24.70 (30)	399.03 ± 22.01 (30)	383.50 ± 16.61* (30)	380.50 ± 26.00* (30)
35	432.97 ± 28.80 (30)	434.10 ± 25.73 (30)	415.70 ± 17.76* (30)	410.20 ± 28.85* (30)
42	455.80 ± 29.91 (30)	461.00 ± 27.45 (30)	441.13 ± 20.38 (30)	432.40 ± 30.92* (30)
49	481.57 ± 34.51 (30)	486.27 ± 30.25 (30)	464.50 ± 22.36 (30)	456.77 ± 34.62* (30)
56	507.40 ± 38.10 (30)	509.43 ± 30.84 (30)	486.73 ± 25.76* (30)	477.63 ± 37.22* (30)
63	522.40 ± 40.78 (30)	528.83 ± 34.29 (30)	504.53 ± 27.53 (30)	495.13 ± 38.69* (30)
70	546.90 ± 42.46 (30)	549.47 ± 39.14 (30)	524.63 ± 29.06 (30)	509.77 ± 41.21* (30)

$ Numbers in parenthesis ( ) equal number of animals used in mean

* Significantly different from Controls at P ≤ 0.05

 

Table 5. Summary of P0 Male Mating Phase (77-98) and Post-Mating Phase (105-141,142) Body Weights(grams) (Mean ± SD)

Study days	Dietary level ppm
	Control (0)	10	750	1500
77	545.73 ± 44.19 (30)$	547.50 ± 38.86 (30)	521.57 ± 30.25* (30)	511.60 ± 42.24* (30)
84	558.70 ± 46.35 (30)	559.07 ± 40.86 (30)	533.50 ± 29.38* (30)	522.57 ± 43.81* (30)
91	579.87 ± 49.25 (30)	578.87 ± 45.55 (30)	553.63 ± 32.41 (30)	539.73 ± 46.92* (30)
98	590.23 ± 51.84 (30)	592.33 ± 47.41 (30)	566.83 ± 34.14 (30)	551.23 ± 49.44* (30)
105	602.13 ± 53.35 (30)	605.87 ± 45.29 (30)	578.90 ± 34.90 (30)	561.10 ± 49.39* (30)
112	613.23 ± 56.41 (30)	616.73 ± 47.69 (30)	585.70 ± 37.43 (30)	569.30 ± 50.76* (30)
119	618.90 ± 57.37 (30)	622.23 ± 48.89 (30)	592.50 ± 38.41 (30)	572.47 ± 52.20* (30)
126	635.90 ± 56.05 (30)	637.40 ± 48.05 (30)	606.03 ± 37.47 (30)	583.87 ± 52.06* (30)
133	646.87 ± 60.95 (30)	643.97 + 52.01 (30)	615.00 ± 40.78 (30)	596.63 ± 55.88* (30)
TBW ^	631.27 ± 60.92 (30)	632.27 ± 52.45 (30)	601.57 ± 41.04 (30)	583.73 ± 55.13* (30)

$ Numbers in parenthesis ( ) equal number of animals used in mean

^ TBW = Terminal body weight – Day 141 or 142 of study

* Significantly different from Controls at P ≤ 0.05

 

Table 6. Summary of P0 Female Pre-Mating Body Weights (grams) (Mean ± SD)

Study days	Dietary level ppm
	Control (0)	10	750	1500
0	167.37 ± 14.63 (30)$	169.07 ± 11.52 (30)	169.97 ± 11.47 (30)	170.90 ± 9.32 (30)
7	189.60 ± 17.18 (30)	190.37 ± 14.89 (30)	187.83 ± 13.35 (30)	184.23 ± 10.62 (30)
14	212.63 ± 21.27 (30)	211.53 ± 17.51 (30)	213.13 ± 15.83 (30)	206.40 ± 12.19 (30)
21	225.40 ± 23.45 (30)	227.27 ± 17.88 (30)	228.73 ± 16.27 (30)	220.23 ± 11.88 (30)
28	238.97 ± 26.81 (30)	241.07 ± 22.10 (30)	241.20 ± 18.84 (30)	229.77 ± 15.05 (30)
35	249.00 ± 29.07 (30)	253.77 ± 24.34 (30)	254.50 ± 22.58 (30)	243.10 ± 15.03 (30)
42	259.30 ± 29.45 (30)	263.27 ± 26.79 (30)	264.80 ± 25.04 (30)	250.03 ± 16.57 (30)
49	269.20 ± 32.53 (30)	276.20 ± 29.09 (30)	275.17± 27.30 (30)	254.73 ± 17.78 (30)
56	278.67 ± 37.06 (30)	283.97 ± 30.39 (30)	282.83 ± 29.90 (30)	261.87 ± 16.60 (30)
63	288.60 + 39.57 (30)	293.43 ± 34.19 (30)	290.47 ± 30.56 (30)	269.90 ± 19.96 (30)
70	298.63 ± 41.69 (30)	299.27 ± 33.96 (30)	298.87 ± 33.64 (30)	278.03 ± 20.99* (30)

$ Numbers in parenthesis ( ) equal number of animals used in mean

* Significantly different from Controls at P ≤ 0.05

 

Table 7. Summary of P0 Female Gestation Body Weights (grams) (Mean ± SD)

Gestation Day	Dietary level ppm
	Control (0)	10	750	1500
0	307.90 ± 41.66 (29)$	306.29 ± 35.44 (24)	301.59 ± 25.50 (29)	283.31 ± 20.26* (29)
7	335.03 ± 44.18 (29)	339.71 ± 31.23 (24)	328.76 ± 27.87 (29)	308.10 ± 21.93* (29)
14	362.52 ± 46.27 (29)	370.92 ± 32.83 (24)	359.10 ± 28.55 (29)	336.83 ± 21.00* (29)
20	431.69 ± 52.65 (29)	446.54 ± 30.84 (24)	427.14 ± 39.66 (28)	400.62 ± 30.23* (29)

$ Numbers in parenthesis ( ) equal number of animals used in mean

* Significantly different from Controls at P ≤ 0.05

 

Table 8. Summary of P0 Female Lactation Body Weights (grams) (Mean ± SD)

Gestation Day	Dietary level ppm
	Control (0)	10	750	1500
0	339.34 ± 45.87 (29)$	347.04 ± 35.32 (26)	333.11 ± 29.88 (28)	308.34 ± 23.15* (29)
7	345.52 ± 40.34 (29)	354.38 ± 27.15 (26)	346.26 ± 25.92 (27)	328.90 + 20.94 (29)
14	358.76 ± 35.16 (29)	370.15 ± 29.11 (26)	358.78 + 23.74 (27)	336.24 ± 23.50* (29)
20	342.83 ± 31.92 (29)	349.65 ± 28.28 (26)	350.22 ± 20.85 (27)	340.72 ± 17.8 (29)

$ Numbers in parenthesis ( ) equal number of animals used in mean

* Significantly different from Controls at P ≤ 0.05

Table 9. Summary of F0 (P1) generation precoital interval.

Dietary level (ppm)	Percent of females mating during cohabitation days
	1 – 4	5 – 8	9 – 12	13 – 16	17 – 21
0	82.8 (24/29)a, b	6.9 (2/29)	3.4 (1/29)	0 (0/29)	6.9 (2/29)
10	90 (27/30)c	3.3 (1/30)	0 (0/30)	3.3 (1/30)	3.3 (1/30)
750	93.3 (28/30)	6.7 (2/30)	0 (0/30)	0 (0/30)	0 (0/30)
1500	96.7 (29/30)	0 (0/30)	0 (0/30)	3.3 (1/30)	0 (0/30)

a) Numbers in oarentheses ( ) represents the number of females with evidence of mating during that interval over the total number of females in that group showing evidence of mating during the entire mating period

b) 0 ppm – one animal sacrificed after 1 day of mating

c) 10 ppm – no sperm was observed in two females. The mating date was estimated by subtracting 23 days from the date of pup delivery

 

Table 10. Summary of F0 generation reproductive parameters (mean and standard deviation)(a)

	Dietary level (ppm)
Parameter	Control (0)	10	750	1500
Number of pregnant females	29	26	29	29
Number of viable litters	29	26	28	29
Number of implantation sites	15.97 ± 2.26	15.92 ± 2.37	16.66 ± 2.24	16.66 ± 1.76
Number of stillbirths	0.31 ± 0.71	0.31 ± 0.79	0.11 ± 0.31	0.38 ± 1.01
% stillbirths	2.21 ± 4.74	2.06 ± 5.34	0.68 ± 2.01	2.43 ± 6.41
Number of viable newborns	14.21 ± 2.11	14.81 ± 2.81	15.25 ± 2.17	15.24 ± 2.03
Post-implantation loss (b)	1.76 ± 1.33	1.15 ± 1.08	1.61 ± 1.37	1.45 ± 1.66
% post-implantation loss (b)	10.85 ± 7.83	7.74 ± 8.25	9.42 ± 8.41	8.44 ± 9.42

a) Although means and standard deviations are reported, the actual statistical analysis is based on non-parametric methods

b) When the number of viable pups exceeds the number of implants, the post-implantation loss was calculated as zero to ensure meaningful biological interpretation of statistical analysis

 

Table 11. Summary of the F0 Generation Reproductive and Fertility Indices (c)

Parameter	Dietary level (ppm)
	Control (0)	10	750	1500
Female fertility	29/29 (a) (100)	26/30 (b) (86.7)	29/30 (96.7)	29/30 (96.7)
Female mating index	29/29 (a) (100)	30/30 (b) (100)	30/30 (100)	30/30 (100)
Gestation index	29/29 (a) (100)	26/26 (100)	28/29 (96.6)	29/29 (100)
Male fertility	29/29 (a) (100)	26/30 (b) (86.7)	29/30 (96.7)	29/30 (96.7)
Male mating index	29/29 (a) (100)	30/30 (b) (100)	30/30 (100)	30/30 (100)

a) One female sacrificed on study day 75 after 1 day of mating.

b) Two females were not observed as sperm positive but delivered litters

c) Data expressed as group ratios and percentages (in parenthesis)

 

Table 12. Summary of the F1 Litter size (Day 0 lactation) survival indices and sex ratios

Parameter	Dietary level (ppm)
	Control (0)	10	750	1500
Number of litters born	29	26	28	29
Mean litter size (excluding stillbirths)	14.21	14.81	15.25	15.24
Mean number of stillbirths	0.31	0.31	0.11	0.38
Number of viable males (Day 0)	223	183	213	230
Number of viable females (Day 0)	189	202	214	212
Survival indices Sexes pooled
Mean % pups surviving, days 0 – 4 (pre-cull)	99.35	94.64	95.13	97.31
Mean % pups surviving, days 4 – 21 (post-cull)	99.14	99.04	99.34	99.55
Survival indices by sex
Mean % males surviving, Days 0 – 4 (pre-cull)	98.99	96.05	95.39	97.51
Mean % males surviving, Days 4 - 21 (post-cull)	99.14	99.04	99.07	99.11
Mean % females surviving, Days 0 – 4 (pre-cull)	99.43	93.83	95.11	97.21
Mean % females surviving, Days 4 - 21 (post-cull)	99.14	99.04	100	100
Sex ratio Day 0 Lactation	54.13	47.53	49.88	52.04

 

Table 13. Summary of F1 male pup body weights (grams, (a) mean and standard error)

Postnatal days	Dietary levels (ppm)
	Control (0)	10	750	1500
0	6.34 ± 0.09 (28)(b)	6.38 ± 0.10 (26)	6.18 ± 0.09 (28)	6.05 ± 0.09 *
4 (pre-culling)	9.53 ± 0.21 (29)	9.57 ± 0.23 (26)	9.02 ± 0.22 (27)	8.9 ± 0.21 *
4 (post-culling)	9.45 ± 0.22 (29)	9.7 ± 0.23 (26)	8.99 ± 0.22 (27)	8.85 ± 0.22 (29)
7	15.73 ± 0.36 (29)	15.99 ± 0.38 (26)	14.94 ± 0.37	14.62 ± 0.36 *
14	33.35 ± 0.56 (29)	33.56 ± 0.59 (26)	31.66 ± 0.58 *	30.22 ± 0.56 *
21	55.03 ±1 (29)	55.96 ± 1.06 (26)	51.44 ± 1.04 * (27)	48.46 ± 1.02 * (29)

a) Values for the weighted means and standard errors are derived from the Healy analysis

b) Numbers in parenthesis ( ) equal number of litters used in mean

* Statistically significant at P ≤ 0.05.

 

Table 14. Summary of F1 female pup body weights (grams, (a), mean and standard error)

Postnatal days	Dietary levels (ppm)
	Control (0)	10	750	1500
0	5.98 ± 0.09 (28) (b)	6.09 ± 0.1 (26)	5.83 ± 0.09 (28)	5.68 ± 0.09 * (28)
4 (pre-culling)	9.13 ± 0.2 (29)	9.25 ± 0.21 (26)	8.66 ± 0.21 (27)	8.44 ± 0.2 * (29)
4 (post-culling)	9.14 ± 0.2 (29)	9.32 ± 0.22 (26)	8.72 ± 0.21 (27)	8.44 ± 0.2 * (29)
7	15.26 ± 0.34 (29)	15.18 ± 0.36 (26)	14.43 ± 0.35 (27)	13.87 ± 0.34 * (29)
14	32.25 ± 0.56 (29)	32.17 ± 0.59 (26)	30.58 ± 0.58 * (27)	29.04 ± 0.56 * (29)
21	52.91 ± 0.94 (29)	53.27 ± 1 (26)	49.32 ± 0.97 * (27)	47.02 ± 0.94 (29)

a) Values for the weighted means and standard errors are derived from the Healy analysis

b) Numbers in parenthesis ( ) equal number of litters used in mean

* Statistically significant at P ≤ 0.05.

 

Table 15. Summary of mean F1 litter size and weight (a)

Dietary level	0		4 (PR) (b)		4 (P0) (c)		7		14		21
	Size	Weight	Size	Weight	Size	Weight	Size	Weight	Size	Weight	Size	Weight
0	14.21	6.18	14.10	9.34	8	9.3	7.93	15.50	7.93	32.81	7.93	53.98
10	14.81	6.25	14	9.44	7.81	9.52	7.73	15.60	7.73	32.87	7.73	54.67
750	15.25	6.01	15.26	8.86	8	8.86	7.96	14.68	7.96	31.11	7.96	50.36
1500	15.18	5.88	14.79	8.69	8	8.64	8	14.24	7.97	29.62	7.83	47.9

a) Size – mean number of viable pups per viable litter; weight – mean pup weight (g) per viable litter

b) PR – pre-culling

c) P0 – post-culling

 

Table 16. Summary of F1 Female Gestation Food Consumption (g/day), mean and SD.

Interval	Study days	Dietary levels (ppm)
		0	10	750	1500
1	0 – 7	21.94 ± 2.67 (21)	21.40 ± 1.91 (23)	20.37 ± 2.84 (28)	20.04 ± 1.58 * (24)
2	7 – 14	23.05 ± 2.68 (21)	23.36 ± 2.25 (23)	22.56 ± 2.83 (28)	21.47 ± 1.46 (24)
3	14 – 20	25.26 ± 2.77 (21)	25.73 ± 2.37 (23)	24.86 ± 3.47 (28)	23.45 ± 1.67 (24)

Number in parenthesis ( ) equal number of animals used in mean.

* Significantly different from Controls at P ≤ 0.05

 

Table 17. Summary of F1 (P2) generation precoital interval.

Dietary level (ppm)	Percent of females mating during cohabitation days
	1-4	5-8	9-12	13-16	17-21
0	92 (23/25^b)^a	0	0	4 (1/25)	4 (1/25)
10	82.1 (23/28)	3.6 (1/28)	0	3.6 (1/28)	10.71 (3/28)
750	86.7 (26/30)	0	0	10 (3/30)	3.3 (1/30)
1500	93.3 (28/30)	3.3 (1/30)	0	0	3.3 (1/30)

(a) Number in parenthesis represents the number of females with evidence of mating during that interval over the total number of females in that group showing evidence of mating during the entire mating period.

(b) In one female sperm was not observed in the vaginal washings; however she delivered a viable litter

 

Table 18. Summary of F1 generation reproductive parameters (mean ± standard derivation)

Parameter	Dietary level (ppm)
	Control (0)	10	750	1500
Number of pregnant females	22	23	28	24
Number of viable litters	21	20	27	24
Number of implantation sites	14.4 ± 4.57	11.83 ± 5.7	15.50 ± 3.54*	16.75 ± 3.98*
Number of stillbirths	0.18 ± 0.5	0.23 ± 0.53	0.37 ± 1.28	0.21 ± 0.41
% stillbirths	1.64 ± 4.38	1.74 ± 3.99	2.07 ± 6.76	1.33 ± 2.75
Number of viable newborns	12.05 ± 4.98	10.82 ± 5.68	14.22 ± 3.48*	14.83 ± 3.58*
Post-implantation loss (a)	2.19 ± 2.6 (b)	1 ± 1.2	1.48 ± 1.81	1.92 ± 2.8
% Post-implantation loss	18.96 ± 24.90	15.97 ± 28.47	9.09 ± 10.68	10.83 ± 13.08

(a) When the number of viable pups and stillbirths exceeded the number of implants, the post-implantation loss was calculated as zero to ensure meaningful biological interpretation of the statistical analysis.

* Statistically different at P ≤ 0.05

 

Table 19. Summary of F1 generation reproductive and fertility indices.

Parameter	Dietary level – ppm
	Control (0)	10	750	1500
Female fertility	22/25 (88)	23/28 (82.1)	28/30 (93.3)	24/30 (80)
Female mating index	25/30 (83.3)	28/30 (93.3)	30/30 (100)	30/30 (100)
Gestation index	21/22 (95.5)	20/23 (87)	27/28 (96.4)	24/24 (100)
Male fertility	22/25 (88)	23/28 (82.1)	28/30 (93.3)	24/30 (80)
Male mating index	25/30 (83.3)	28/30 (93.3)	30/30 (100)	30/30 (100)

 

Table 20. Summary of F2 litter size (Day 0 lactation). Survival indices and sex ratios.

Parameter	Dietary level (ppm)
	Control (0)	10	750	1500
Number of litters born	21	20	27	24
Mean litter size (day 0 lactation; excluding stillbirths)	12.62	11.9*	14.22*	14.83*
Mean no. stillbirths	0.18	0.23	0.37	0.21
Number of viable males (day 0)	126	112	195	167
Number of viable females (day 0)	139	126	189	189
Survival indices sexes pooled:
Mean % pups surviving, Days 0-4 (pre-cull)	99.68	93.66	96.86	96.55
Mean % pups surviving, Days 4-21 (post-cull)	99.38	97.37	100	98.96
Survival Indices by sex:
Mean % males surviving, Day 0-4 (pre-cull)	100	93.11	96.08	96.47
Mean % males surviving, Days 4-21 (post-cull)	98.68	98.68	100	100
Mean % females surviving, Day 0-4 (pre-cull)	99.38	94.55	97.60	96.05
Mean % females surviving, Days 4-21 (post-cull)	100	96.05	100	98.26
Sex ratio Day 0 Lactation (% males)	47.55	47.06	50.78	46.91

 

Table 21. Summary of mean F2 litter size and weight (day of lactation).

Dietary level (ppm)	0		4 (PR)		4 (PO)		7		14		21
	Size	Weight	Size	Weight	Size	Weight	Size	Weight	Size	Weight	Size	Weight
0	12.62	6.4	12.57	9.5	7.33	9.47	7.3	16.68	7.29	30.44	7.4	48.96
10	11.9	6.48	12.05	9.13	7.37	9.17	7.26	16.34	7.16	30.37	7.16	49.51
750	14.22	6.04	13.7	8.92	7.85	8.95	7.85	16.27	7.85	29.26	7.85	46.96
1500	14.83	5.97	14.17	8.48	7.83	8.44	7.79	15.09	7.79	27.26	7.75	43.81

Conclusions:

The lowest NOAEL for parental, systemic toxicity was considered to be 10 ppm for the P1 generation males (0.48 - 0.93 mg/kg bw/day) and 750 ppm for the P1 generation females (43.66 - 76.04 mg/kg bw/day).
The NOAEL for reproduction toxicity was considered to be in excess of 1500 ppm for P0 and P1 generation males and females (corresponding to average doses of 99.6 mg/kg bw/day in males and 105.0 mg/kg bw/day in females).
The NOAEL for foetal toxicity was considered to be 750 ppm for F1 and F2 generations (corresponding to average doses of 53.1 mg/kg bw/day in males and 53.6 mg/kg bw/day in females).

Executive summary:

In this two-generation reproductive toxicity study (GLP and EPA 83-4) the test substance was administered in the diet to three groups of male and female rats at concentrations of 10, 750, or 1500 ppm for two successive generations (P0, P1). A fourth group of rats received diet without the test substance for two generations and served as controls. There were no compound-related mortalities or clinical observations during treatment of either parental (P0 or P1) generation. Reductions in mean body weight parameters were observed in the 1500 ppm P0 and the 750 ppm and 1500 ppm P1 males and females during the premating period and frequently persisted into gestation and lactation for females. Treatment-related reductions in food consumption were noted at 1500 ppm of the P0 generation and at 750 ppm and 1500 ppm in the P1 generation for both sexes during premating. P0 and P1 female food consumption during gestation was also reduced at 1500 ppm. Mating and fertility indices were comparable for the control and treated groups of both generations. There were no treatment-related effects on the duration of gestation, fertility indices, or any of the reproductive parameters. Mean ovary and testes weights were comparable among treated and control groups. The mean relative testes weight was significantly increased at the 1500 ppm dose as a result of the reduced mean terminal body weight in this group. Necropsy and histopathological examinations of the P0 and P1 parental generation animals and selected F2 pups did not reveal any effects attributable to treatment. Litter size, sex ratio, and postnatal survival indices of the F1 and F2 generation were unaffected by treatment. There were no treatment-related clinical observations, necropsy observations, or malformations. The 750 ppm and 1500 ppm F1 and the 1500 ppm F2 pup weights were reduced at most intervals during lactation. The lower pup weights were influenced by a slightly larger litter size, maternal toxicity, and ingestion of the test substance by the pups. Pups were similar to controls at 10 ppm.
The study data indicate that the test substance did not cause any adverse effects on the overall reproductive performance in rats fed concentrations up to and including 1500 ppm for two generations. Under the conditions of this study, the no observable effect level (NOEL) was 10 ppm.

The NOAEL for systemic toxicity was considered to be 750 ppm for P0 males and females (on average 53.1 and 53.6 mg/kg bw/day, respectively) and 10 ppm for the P1 generation males (on average 0.68 mg/kg bw/day) and 750 ppm for the P1 generation females (on average 59.9 mg/kg bw/day). The NOAEL for reproduction toxicity was considered to be in excess of 1500 ppm for P0 and P1 generation males and females (99.6 mg/kg bw/day in males and 105.0 mg/kg bw/day in males and females, respectively). The NOAEL for feotal toxicity was considered to be 750 ppm for F1 and F2 generations (53.1 mg/kg bw/day in males and 53.6 mg/kg bw/day in females, respectively).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

53.1 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP compliant 2-generation study, EPA 83-4

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

EPA 83-4 in rats (Giknis 1990)
The reproductive toxicity to the rat was studied in a GLP compliant two-generation study to EPA 83-4 in which the test substance was administered in the diet to male and female rats at concentrations of 10, 750, or 1500 ppm for two successive generations. The control group received plain diet without the test substance for two generations. The lowest NOAEL for parental systemic toxicity of 10 ppm or 0.68 mg/kg bw/day was established for P1 males. There were no treatment-related effects on any reproductive parameters examined through both generations. No impairment of testes or ovary weights of parental animals which was attributable to treatment was noted. There were no treatment-related clinical observations, necropsy findings or malformations in pups of either generation. Pup weights at intermediate and top dose levels were decreased during lactation. Necropsy and histopathology examination of animals of either generation revealed no treatment-related effects on reproductive organs. The lowest NOAEL for reproduction toxicity was 99.6 mg/kg bw/day in P1 males and 105.0 mg/kg bw/day in P1 females; and the lowest NOAEL for foetal toxicity was 53.1 mg/kg bw/day in F1 males and 53.6 mg/kg bw/day in F1 females.

Effects on developmental toxicity

Description of key information

The maternal and foetal no observable effect level was 50 mg/kg/day in the rat (Weissenborn 1987)

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Initiation: 22 July 1985; end: 16 August 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

1981

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Source: HARE, Maryland, Hewitt, Ney Jersey, USA
Age: not reported
Weight: 2.97 to 4.01 kg
Acclimation period: 4 weeks
Housing: rabbits were caged individually in mesh bottom stainless steel cages that were changed bi-weekly
Temperature: 18 ± 3 °C
Humidity: 50 ± 20%
Lighting: 14 hours light to 10 hours darkness cycles
Food: Purina Certified Rabbit Chow ad libitum
Water: ad libitum

Route of administration:

oral: gavage

Vehicle:

other: Test substance was suspended in 3% cornstarch containing 0.5% Tween 80

Details on exposure:

Substance was administered once daily by oral gavage as 0.04%, 0.24% or 1.44% suspension in the vehicle. A volume of 5 mL/kg bw/day was used. Exposure occurred from day 7 through day 19 of gestation.

Analytical verification of doses or concentrations:

no

Details on mating procedure:

Sexually mature virgin female rabbits were artificially inseminated using semen collected from the buck colony of the same strain maintained at the testing facility.

Duration of treatment / exposure:

Day 7 through day 19 of gestation

Frequency of treatment:

Daily treatment by oral gavage

Duration of test:

29 days

Dose / conc.:

2 mg/kg bw/day (nominal)

Dose / conc.:

12 mg/kg bw/day (nominal)

Dose / conc.:

72 mg/kg bw/day (nominal)

No. of animals per sex per dose:

19 females

Control animals:

yes, concurrent vehicle

Details on study design:

A computer program was used to randomly distribute 76 females into four groups of 19 animals each.

Maternal examinations:

The does were observed daily for changes in general appearance and behaviour. Mortality was checked twice daily. Females were weighed on days 0, 7, 10, 14, 20, 24 and 29 of gestation. Feed consumption measurements were taken daily for gestational day 5 through 28. At the time of necropsy, all does were examined for gross pathologic changes. Maternal gross lesions were excised, stored in formalin and eventually evaluated under the microscope.

Ovaries and uterine content:

Not described in report: number of corpora lutea, implantations, viable foetuses, resorptions was recorded for each doe

Blood sampling:

No

Fetal examinations:

At necropsy each foetus was examined viscerally and its sex determined. This examination included the brain, heart, major blood vessels, trachea, lungs, diaphragm, oral cavity, tongue, esophagus, stomach, intestines, liver, gall-bladder, pancreas, thymus, spleen, kidneys, ureters, bladder, adrenals, ovaries and uterus or testicles.
Following the visceral examination the foetuses were placed in 95% ethanol, stained with Alizarin Red S and prepared for skeletal examination. All ossification centres characteristically present at day 29 of gestation in this rabbit strain were checked for presence or absence, size, shape, location and relationship to adjacent ossification centres.

Statistics:

Statistical analysis of the data were performed. Parametric analysis was performed on body weight, weight gain and feed consumption using Bartlett's Test for homogeneity of variance. For homogeneous variances, one-way analysis of variance with Dunnett's method of multiple comparisons was used. Parametric analysis of foetal weight was done using Healy analysis. Non-parametric analysis was performed on the number of corpora lutea, implantations, viable foetuses, resorptions and percentage of post-implantation loss.

Indices:

% post-implantation loss was calculated as follows: [(number of implantation sites - number of viable foetuses)/number of implantation sites] x 100 per doe

Historical control data:

No data provided

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Few clinical signs were noted for all groups, including alopecia and decreased, soft or no stool. Blood in the pan or vaginal blood was observed in 2 females receiving 12 mg/kg bw/day and 4 females receiving 72 mg/kg bw/day. Other clinical signs were occurring sporadically.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Three females died during the course of the study, two from the high-dose and one from the mid-dose group. One female died as a result of a dosing accident. Post-mortem examination of a second female revealed the presence of a hair ball in the stomach that was most likely the cause of the death. No explanation could be found for the death of the third animal. Blood was found in the cage pan, and this female may have been aborting. Since no signs of toxicity were observed prior to death, this death was not related to the treatment. An additional three animals were sacrificed during the course of the study due to abortion or early delivery.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant reductions in weight gain were detected in the group dosed at 72 mg/kg bw for the test intervals from day 10 to 14 and day 14 to 20 of gestation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A statistically significant reduction in the feed consumption was observed in females dosed with 72 mg/kg bw/day.

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Nine females in the study were observed to have abnormal findings at necropsy that were not attributed to treatment. One female in the control group had pitted kidneys. In the group dosed with 12 mg/kg bw/day, one female was observed to have cream coloured amniotic fluid surrounding a foetus, one had a trilobate spleen probably due to an event in early life not related to treatment, and one had a gallbladder that was reduced in size. Two females in the high-dose group were necropsied due to unscheduled death: a hair ball was found in the stomach of one animal, while the second died following a dosing accident. One female presented with a cavitation measuring about 4 mm in diameter in the region of the renal cortex. Another female presented with an enlarged spleen and one with a gallbladder with an irregular, bumpy surface.

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

An increase in the mean number of resorptions (early, late and total) and resulting increased post-implantation loss was observed in the group dose at 72 mg/kg bw/day, though it was not statistically significant. This observation was due to females whose pregnancies did not produce viable foetuses.

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

12 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Two live foetuses were observed to have external malformations. A control foetus had ascites and a foetus from the high-dose group was noted as having a domed cranium. A dead foetus from the high-dose group was found to have a domed cranium filled with green fluid.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One foetus from the high-dose group was found to have an additional rib and sternebrae. A statistically significant incidence of forepaw proximal phalanges not completely ossified was observed in two low birthweight foetuses from two litters in the high-dose group. This finding was linked to the maternal toxicity observed in this group and thought to represent a slight delay in normal development rather than direct foetotoxicity.

Visceral malformations:

no effects observed

Description (incidence and severity):

Very few visceral malformations were observed (including hydrocephalus, ovarian cyst, misshapened heart, shortened renal papillae, fissures on the dorsal lung surface, elongated median liver lobe, agenesis of the gallbladder) and considered to be spontaneous in nature.

Key result

Dose descriptor:

NOAEL

Effect level:

72 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

Despite signs of maternal toxicity, the substance was not embryotoxic, foetotoxic, or teratogenic at doses as high as 72 mg/kg bw/day.

Executive summary:

The potential developmental toxicity of the substance to rabbits was studied under GLP to OECD TG 414. Technical grade material was suspended in 3% corn starch containing 0.5% of Tween 80 and administered by gavage at daily doses of 2, 12, and 72 mg/kg to New Zealand White rabbits (19 animals/group) from days 7 through 19 of gestation. The vehicle not containing test substance was administered to a separate group of 19 pregnant female rabbits serving as the negative control group. Treatment-related effects of maternal toxicity were observed only at the high dose level, including reduction in feed consumption and in maternal body weight gain. In addition, vaginal bleeding was noticed. There were no treatment-related deaths in this study nor any adverse effects on any of the reproductive parameters examined, on foetal body weight, foetal sex ratios or on the incidence of foetal gross, visceral, or skeletal malformations. In two high-dose group foetuses a statistically significant increase in the incidence of a minor skeletal variation was noticed (incompletely ossified forepaw proximal phalanges). This variation was considered to be attributable to the observed maternal toxicity and thought to represent a slight delay in normal development rather than direct foetotoxicity.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 Nov 1986 to 05 Dec 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 418

Version / remarks:

May 12, 1981

Qualifier:

according to guideline

Guideline:

other: Proposed Health Effects Test Standards for Toxic Substances Control Act Test Rules, US EPA, Red. Reg., Vol. 44, No.91

Version / remarks:

1979

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl: COBS CD (SD) BR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Sexually mature
- Weight at study initiation: 201-274 g (females), 453-744 g (males)
- Housing: Individually, except during mating, in solid bottom cages cotnaining hard-wood chips which were changed once a week.
- Diet: Certified Chow ad libitum
- Water: Tap water ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 26
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 10/14

IN-LIFE DATES: 17 Nov 1986 to 05 Dec 1986

Route of administration:

oral: gavage

Vehicle:

other: 3% aqueous corn starch with 0.5% Tween

Details on exposure:

VEHICLE
- Concentration in vehicle: 0.1, 0.5 or 2.5%
- Amount of vehicle: 10 mL/kg bw/day
- The test article, as formulated in the control article was stable for 24 hours at room temperature and at least 33 days at 6°C.
- The vehicle was stable for at least 36 days from the date of manufacture when stored as directed.

DOSING
Due to a techinal error, one female (Group 2) was not dosed on day 6 of gestation an one female (Group 4) was overdosed by 0.63 mL on day 14 of gestation. These misdosings were considered to have no adverse effects on the quality, integrity or data interpretation of this study.

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

140 sexually mature virgin female rats were mated with 60 sexually mature males. Conformation of mating was determined by the presence of sperm in the vaginal washing. Sperm in vaginal was referred to as day 0 of pregnancy.

Duration of treatment / exposure:

From day 6 to day 15 of gestation

Frequency of treatment:

Daily

Duration of test:

Until day 20 of gestation

Dose / conc.:

10 mg/kg bw/day

Remarks:

Group 2, low dose

Dose / conc.:

50 mg/kg bw/day

Remarks:

Group 3, intermediate dose

Dose / conc.:

250 mg/kg bw/day

Remarks:

Group 4, high dose

No. of animals per sex per dose:

26

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS:
The dams were observed twice daily for changes in appearance or behaviour

BODY WEIGHT:
Body weights were recorded on gestational days 0, 6-16, 20.

FOOD CONSUMPTION
Feed consumption was recorded for a weekly period from day 0 tot day 6, and daily thereafter throughout gestation.

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day # 20
- All dams were examined grossly. No specimens were submitted to the Pathology Section for microscopic evaluations.

Ovaries and uterine content:

The uteri, including their contents, with ovaries attached, were weighed and the corpora lutea, live foetuses, dead foetuses and intrauterine resorption sites were counted. The foetuses were sexed and numbered in order of their positions in the uterus from the ovarian end of the left horn to the ovarian end of the right horn. Viable foetuses were weighed and examined for gross abnormalities, including inspection of the palate. Each foetus was tagged with the live foetus number as designated by its position in the uterus. Approximately one half of the foetuses from each litter were placed into Bouin's fixative for subsequent visceral examination and the remaining half of the litter was placed in 95% ethanol for subsequent staining and skeletal examination.

Fetal examinations:

EXTERNAL EXAMINATIONS AND SOFT TISSUE EXAMINATIONS
Approximately one half of the fetuses were fixed in Bouin's solution for at least one week and then examined for visceral abnormalities according to the method of Monie, Kho, and Morgan (1965). The following systems and organs were examined: Central Nervous System (brain, eyes, spinal cord), Cardiovascular system (heart, major blood vessels), Respiratory System (nasal passages, trachea, lungs, diaphragm), Gastrointestinal System (oral cavity, tongue, stomach, oesophagus, intestines, liver, pancreas), Lymphoid Structures (thymus, spleen), Urinary System (kidneys, ureters, bladder), Endocrine System (adrenals), Reproductive System (ovaries, uterus or testicles)

SKELETAL EXAMINATIONS
Approximately one half of foetuses from each litter were stained with Alizarin Red S, cleared according to the method of Staples and Schnell (1964), and subsequently examined for skeletal abnormalities.
The rodent skeletal examination involves checking, with the aid of appropriate magnification, all ossification centres that are characteristic of a rat foetus on gestational day 20 (Walker & Wirtschafter, 1967). The examination involves checking for their presence/absence, size, shape, location and relationship to adjacent ossification centres.

Statistics:

Statistical analyses of all data were performed as follows, unless otherwise specified:
- Body Weight, Body Weight Gain and Feed consumption: One-Way Analysis of Variance (ANOVA, Snedecor and Cochran, 1968), Bartlett’ s Test for Homogeneity of Variance (Snedeco and Cochran, 1968) and Dunnett’ s Method of Multiple Comparisons between control and treatment groups (Dunnett, 1955, 1964). Statistically significant differences between treatment groups are not discussed.
- Foetal weight: Healy Analysis (Healy, 1972)

Indices:

Post-implantation loss = (No. of Implantation Sites) - (No. of Viable Fetuses) / (No. of Implantation Sites) x 100%

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The only clinical observation was salivation which was oberverd in 4 of 26 high-dose dams.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

All dams survived to scheduled sacrifice on day 20 of gestation.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight was not recorded for one female (low-dose) on day 6 of gestation due to a technical error. However, this incident had no adverse effect on the outcome of this study or the interpretation of the data.
There were no compound-related effects on maternal body weight or body weight gain prior to the initiation of dosing on gestational days 0-6 in any treatment group. Body weights were reduced during the dosing period for dams of both the intermediate and high-dose groups. These reductions were statistically lower than control values (p ≤ 0.05) on gestational days 8, 9 and 10 in the intermediate-dose group; and gestational days 7-16 in the high-dose group. The corrected gestational day 20 body weight (maternal body weight less the weight of the uterus, placentas and foetuses) was also significantly lower than the control value for dams of the high-dose group. The body weight reductions in the high-dose group were attributed to maternal toxicity; however, those at the intermediate-dose level were minimal, transient and not associated with any clinical signs of toxicity or alterations in feed consumption and body weight gain. Therefore, they are considered to be unrelated to treatment. Maternal body weights for the intermediate and high-dose groups were comparable to the control group on gestational day 20, thus indicating maternal recovery in the post-dosing period. Maternal body weights were unaffected in the low-dose group.
Maternal body weight gains and corrected body weight gains did not vary significantly from control values in the low- or intermediate-dose groups at any time in the study. However, statistically significant reductions in body weight gain (or actual body weight losses) were noted in the high- dose group on gestational days 6-8, 8-12, 6-16, and 0-20 and for gestational days 6-7, 7-8, 8-9, 10-11 and 15-16 during the treatment period (see Table 1, 2 and 3 in “Any other information on results incl. tables”). The corrected body weight gain for gestational days 0-20 was also significantly reduced in the high-dose group. The reductions in body weight gain correlate well with the reductions in body weight and food consumption also observed in the high-dose group and are considered to be indicative of compound-related maternal toxicity at the 250 mg/kg/day dose level.
A statistically significant increase in body weight gain as compared to the control group was observed in high-dose dams following cessation of compound administration during the interval of gestational days 16-20. This increase was associated with the increased feed consumption also observed during this period and suggests recovery of dams in the high-dose group during the post-dose period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Feed consumption was not recorded for one female (low-dose) on day 6 of gestation due to a technical error. However, this incident had no adverse effect on the outcome of this study or the interpretation of the data.
There were no statistically significant differences in feed consumption prior to the initiation of dosing in any dose group, or in the low or intermediate dose group at any time during the study (see Table 4 in “Any other information on results incl. tables”). Treatment-related reductions in feed consumption were observed in the high-dose group throughout the dosing period and were statistically significant (p ≤ 0.05) on gestational days 6-7 through day 13-14 and for days 6-16. These reductions were attributed to the maternal toxicity of the test material at the high-dose level and were associated with compound-related reductions in body weight and weight gain in this group. Following the completion of the dosing regimen, feed consumption was slightly increased in the high-dose group. The increase was statistically significant for the intervals of gestational days 18-19 and 16-20 when compared to the control values, and is indicative of maternal recovery in the high-dose group.

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

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no necropsy obervations related to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

The incidences of resorptions, early resorptions and % post-implantation loss were statistically lower than control values for both the intermediate and high-dose group. These differences were considered to be incidental and therefore not related to treatment.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

The incidences of resorptions, early resorptions and % post-implantation loss were statistically lower than control values for both the intermediate and high-dose group. These differences were considered to be incidental and therefore not related to treatment.

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

(=NOEL)

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistical analysis of foetal body weights did not reveal any significant treatment-related changes in either the low or intermediate-dose group as compared to the control group. However, in the high-dose group both male and female foetal weights were significantly reduced (p < 0.05) when compared to control values (see Table 6 in “Any other information on results incl. tables”). These reductions in foetal weight are considered to be secondary to reductions in body weight, body weight gain and feed consumption at the 250 mg/kg/day dose level.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on the foetal sex ratios (see Table 5 in “Any other information on results incl. tables”).

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Description (incidence and severity):

There were no remarkable foetal gross observations.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

A total of 630 foetuses representing 89 litters were given skeletal examinations. The only skeletal malformation observed in this study was rudimentary 13th thoracic ribs which was observed in 1, 9, 3, and 1 foetuses in the control, low-, intermediate- and high-dose group respectively. This malformation was spontaneous in nature and not related to the test material. The majority of these variations generally occurred with similar incidences in the control and treated animals however two minor variations incomplete ossification of the sternebrae and unossified forepaw metacarpals, were observed at a statistically significant (p ≤ 0.05) increased incidence in the high-dose group when the data were analysed on a foetal basis. The increased incidence of these two variations are considered to be secondary to the maternal toxicity observed in the dams of this dose group.

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

A total of 589 foetuses from 89 litters were examined for visceral malformations and variations. Dilated lateral ventricles in the brain were observed in a foetus from the intermediate-dose group and was the only visceral malformation observed in this study. This malformation was not treatment-related.
The only compound-related visceral variation observed in this study was shortened renal papillae which occurred at a statistically significant (p < 0.05) increased incidence in the high-dose group. This effect correlates with the reduced foetal weights observed in the high-dose group and like the reduced foetal weights is most likely secondary to the maternal toxicity observed at this dose level.

Key result

Dose descriptor:

NOAEL

Remarks:

(=NOEL)

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

skeletal malformations

visceral malformations

Key result

Abnormalities:

no effects observed

Localisation:

skeletal: forelimb

skeletal: sternum

visceral/soft tissue: urinary

Description (incidence and severity):

- skeletal: two minor variations incomplete ossification of the sternebrae and unossified forepaw metacarpals, were observed at a statistically significant (p ≤ 0.05) increased incidence in the high-dose group.
- visceral/soft tissue: shortened renal papillae which occurred at a statistically significant (p < 0.05) increased incidence in the high-dose group (54 in the high-dose group compared to 37 in the control group)

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

250 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

no

Table 1. Summary of Maternal Body Weight (grams) (mean±SD)

Days of Gestation	Dose level (mg/kg/day)
	Control (0)	10	50	250
0	239.65 ± 10.24 (23)^	237.08 ± 10.71 (24)	234.70 ± 11.47 (20)	238.00 ± 10.74 (22)
6	274.17 ± 12.50 (23)	269.30 ± 13.12 (23)	264.70 ± 12.44 (20)	270.23 ± 14.50 (22)
7	278.09 ± 16.20 (23)	274.58 ± 11.47 (24)	268.50 ± 12.70 (20)	267.45 ± 12.76* (22)
8	282.74 ± 13.16 (23)	278.96 ± 12.40 (24)	271.90 ± 12.14* (20)	264.27 ± 13.27* (22)
9	288.30 ± 13.47 (23)	282.79 ± 12.59 (24)	276.75 ± 12.17* (20)	265.59 ± 17.04* (22)
10	293.13 ± 14.02 (23)	287.88 ± 11.81 (24)	279.75 ± 12.17* (20)	268.18 ± 16.49* (22)
11	297.83 ± 14.56 (23)	292.67 ± 12.23 (24)	287.45 ± 13.86 (20)	266.68 ± 18.63* (22)
12	301.43 ± 13.86 (23)	296.50 ± 13.00 (24)	292.60 ± 14.31 (20)	271.77 ± 18.46* (22)
13	307.22 ± 16.07 (23)	302.17 ± 16.08 (24)	297.50 ± 14.42 (20)	276.68 ± 22.21* (22)
14	312.83 ± 13.73 (23)	308.75 ± 15.86 (24)	304.45 ± 14.75 (20)	286.27 ± 21.30* (22)
15	319.09 ± 18.35 (23)	315.00 ± 15.01 (24)	309.25 ± 13.64 (20)	295.14 ± 20.58* (22)
16	332.65 ± 17.12 (23)	324.58 ± 15.22 (24)	320.15 ± 14.82 (20)	302.14 ± 22.18* (22)
20	384.39 ± 24.16 (23)	375.96 ± 26.40 (24)	376.75 ± 19.63 (20)	368.14 ± 20.55 (22)
20 U$	307.23 ± 18.99 (23)	300.30 ± 17.79 (24)	299.55 ± 16.57 (20)	292.03 ± 19.55* (22)

^Numbers in parenthesis () equal number of animals used in mean.

$Body weight on day 20 of gestation less the weight of the uterus, ovaries, placentas and foetuses.

* Different from the Control at p≤0.05.

 

Table 2. Summary of Maternal Weight Gain During Specified Intervals (grams) (mean±SD)

Days of Gestation	Dose level (mg/kg/day)
	Control (0)	10	50	250
0-6	34.52 ± 7.64 (23)^	32.00 ± 7.95 (23)	30.00 ± 5.53 (20)	32.23 ± 8.56 (22)
6-8	8.57 ± 5.58 (23)	9.78 ± 3.48 (23)	7.20 ± 3.75 (20)	-5.95 ± 11.11* (22)
8-12	18.70 + 5.32 (23)	17.54 ± 4.65 (24)	20.70 + 5.06 (20)	7.50 ± 13.99* (22)
12-16	31.22 ± 7.42 (23)	28.08 ± 7.91 (24)	27.55 ± 6.76 (20)	30.36 ± 16.99 (22)
6-16	58.48 ± 9.53 (23)	56.04 ± 9.07 (23)	55.45 ± 7.23 (20)	31.91 ± 20.30* (22)
16-20	51.74 ± 10.18 (23)	51.38 ± 16.38 (24)	56.60 ± 10.04 (20)	66.00 ± 14.14* (22)
0-20	144.74 ± 20.40 (23)	138.88 ± 22.41 (24)	142.05 ± 16.71 (20)	130.14 ± 15.00* (22)
0-20 U$	67.61 ± 16.52 (23)	63.25 ± 16.56 (24)	64.85 ± 12.41 (20)	54.00 ± 13.10* (22)

^Numbers in parenthesis () equal number of animals used in mean.

$Day 20 U = Day (20) Body Weight less uterus, ovaries, placentas and foetuses.

* Different from the Control at p≤0.05

 

Table 3. Summary of Daily Maternal Body Weight Gain During Treatment (grams) (mean±SD)

Days of Gestation	Dose level (mg/kg/day)
	Control (0)	10	50	250
6-7	3.91 ± 6.51 (23)^	5.35 ± 3.74 (23)	3.80 ± 3.72 (20)	-2.77 ± 8.74* (22)
7-8	4.65 ± 7.81 (23)	4.38 ± 3.70 (24)	3.40 ± 3.12 (20)	-3.18 ± 5.73* (22)
8-9	5.57 ± 5.13 (23)	3.83 ± 3.19 (24)	4.85 ± 3.22 (20)	1.32 ± 7.26* (22)
9-10	4.83 ± 3.02 (23)	5.08 ± 4.32 (24)	3.00 ± 4.89 (20)	2.59 ± 10.01 (22)
10-11	4.70 ± 3.27 (23)	4.79 ± 4.47 (24)	7.70 ± 5.39 (20)	-1.50 ± 10.14* (22)
11-12	3.61 ± 3.69 (23)	3.83 ± 5.39 (24)	5.15 ± 5.02 (20)	5.09 ± 10.41 (22)
12-13	5.78 ± 4.98 (23)	5.67 ± 7.88 (24)	4.90 ± 4.66 (20)	4.91 ± 11.39 (22)
13-14	5.61 ± 4.43 (23)	6.58 ± 6.95 (24)	6.95 ± 5.50 (20)	9.59 ± 11.53 (22)
14-15	6.26 ± 7.89 (23)	6.25 ± 6.17 (24)	4.80 ± 5.17 (20)	8.86 ± 8.00 (22)
15-16	13.57 ± 6.47 (23)	9.58 ± 6.53 (24)	10.90 ± 4.59 (20)	7.00 ± 8.18* (22)

^Numbers in parenthesis () equal number of animals used in mean.

* Different from the Control at p≤0.05

 

Table 4. Summary of Maternal Feed Consumption (grams/interval) (mean±SD)

Interval	Days of Gestation	Dose level (mg/kg/day)
		Control (0)	10	50	250
1	0-6	136.65 ± 14.85 (23)^	127.78 ± 12.72 (23)	127.45 ± 11.08 (20)	132.36 ± 12.86 (22)
2	6-7	23.96 ± 5.74 (23)	24.30 ± 3.97 (23)	21.90 ± 3.19 (20)	15.14 ± 4.63* (22)
3	7-8	24.74 ± 5.82 (23)	22.96 ± 3.10 (24)	22.30 ± 3.28 (20)	13.36 ± 4.35* (22)
4	8-9	23.13 ± 3.52 (23)	22.33 ± 2.46 (24)	22.85 ± 3.13 (20)	15.59 ± 6.11* (22)
5	9-10	24.74 ± 3.95 (23)	22.92 ± 3.28 (24)	21.89 ± 2.71 (19)	16.59 ± 6.20* (22)
6	10-11	24.55 ± 3.29 (22)	23.63 ± 3.28 (24)	23.10 ± 3.19 (20)	14.77 ± 7.06* (22)
7	11-12	24.48 ± 3.68 (23)	23.29 ± 4.52 (24)	24.70 ± 3.01 (20)	18.23 ± 7.92* (22)
8	12-13	26.91 ± 3.68 (23)	25.04 ± 4.19 (24)	23.85 ± 2.87 (20)	21.41 ± 10.33* (22)
9	13-14	25.70 ± 3.70 (23)	23.67 ± 3.84 (24)	23.70 ± 4.62 (20)	19.91 ± 8.69* (22)
10	14-15	24.00 ± 4.20 (23)	23.71 ± 4.01 (24)	23.40 ± 4.03 (20)	22.95 ± 6.31 (22)
11	15-16	27.83 ± 6.83 (23)	25.04 ± 3.72 (24)	25.80 ± 3.94 (20)	24.23 ± 7.04 (22)
12	16-17	26.22 ± 3.68 (23)	27.42 ± 3.75 (24)	27.25 ± 5.17 (20)	28.50 ± 6.17 (22)
13	17-18	28.39 ± 5.10 (23)	27.58 ± 4.31 (24)	27.90 ± 4.98 (20)	28.91 ± 5.08 (22)
14	18-19	26.22 ± 5.11 (23)	26.71 ± 4.23 (24)	27.75 ± 3.39 (20)	30.91 ± 4.72* (22)
15	19-20	26.39 ± 4.86 (23)	25.46 ± 4.81 (24)	28.50 ± 4.72 (20)	28.95 ± 4.58 (22)
	Pre-treatment 0-6	136.65 ± 14.85 (23)	127.78 ± 12.72 (23)	127.45 ± 11.08 (20)	132.36 ± 12.86 (22)
	Treatment 6-16	250.64 ± 29.90 (22)	237.78 ± 16.91 (23)	232.84 ± 14.67 (19)	182.18 ± 30.81* (22)
	Post-treatment 16-20	107.22 ± 13.12 (23)	107.17 ± 11.80 (24)	111.40 ± 12.47 (20)	117.27 ± 14.61* (22)

^Numbers in parenthesis () equal number of animals used in mean.

* Different from the Control at p≤0.05.

 

Table 5. Summary of Reproductive Parameters (mean±SD)

Parameter	Dose level (mg/kg/day)
	Control (0)	10	50	250
No. Pregnant	23	24	20	22
% Pregnant	88.46	92.31	76.92	84.62
Corpora Lutea $	16.04 ± 1.94	16.83 ± 2.04	16.65 ± 2.66	17.23 + 2.74
Implantation Sites $	14.48 ± 2.02	14.42 ± 3.37	13.90 ± 2.10	15.00 ± 1.45
No. Litters Examined	23	24	20	22
Early Resorptions $	0.87 ± 0.87	1.25 ± 2.25	0.30 ± 0.57*	0.55 ± 0.80*
Late Resorptions $	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00
Resorptions $	0.87 ± 0.87	1.25 ± 2.25	0.30 ± 0.57*	0.55 ± 0.80*
% Post-Implantation Loss $	6.56 ± 8.18	8.48 ± 17.07	2.14 ± 4.10*	3.72 ± 5.74*
Number of Live Foetuses $	13.61 ± 2.48	13.17 ± 3.89	13.60 + 2.14	14.45 ± 1.74
Number of Dead Foetuses $	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00
Foetal Sex Ratio (%males)	50.8	45.2	48.2	53.8

* Different from the Control at p≤0.05

$ Although means and standard deviations are reported, the actual statistical analysis is based on non-parametric methods

 

Table 6. Foetal Weights$ (grams) (mean±SE)

Parameter	Dose level (mg/kg/day)
	Control (0)	10	50	250
Foetal Weight - male	3.61 ± 0.05	3.64 ± 0.05	3.69 ± 0.06	3.37 ± 0.05*
Foetal Weight - female	3.46 ± 0.05	3.44 ± 0.05	3.48 ± 0.05	3.23 ± 0.05*

$ Values for the means and standard errors were obtained from the Healy Test

* Different from the Control at p≤0.05.

Conclusions:

Compound-related maternal effects were observed in the high-dose group and consisted of salivation, decreased feed consumption, body weight and body weight gain with recovery. The test substance had no effect on any of the reproductive parameters examined, foetal sex ratios, or the incidence of gross skeletal or visceral malformations. Foetal toxicity, as indicated by decreased foetal weight and an increased incidence of minor skeletal and visceral variations, was observed at the maternally toxic high-dose level. There was no evidence of embryotoxicity or teratogenicity in this study. The no observable effect level for the dam and the foetus was estimated to be at least 50 mg/kg/day.

Executive summary:

In this study (OECD 418 under GLP), the test substance was evaluated for its embryotoxic, fetotoxic, and teratogenic potential in rats. The test substance was suspended in 3% cornstarch containing 0.5% Tween 80 and administered by gavage at doses of 10, 50, or 250 mg/kg/day to three groups of rats (N=26) on gestational days 6-15. A fourth group of control animals (N=26) received an equivalent volume of the vehicle (10 mL/kg/day). All animals were observed twice daily for clinical signs and mortality. Maternal feed consumption and body weights were recorded during gestation. Following maternal necropsy on day 20 of gestation, the foetuses were weighed and examined for external, visceral and skeletal anomalies. No compound-related effects were observed in the low- or intermediate-dose groups. Signs of treatment related maternal toxicity observed in the high-dose group included salivation and reductions in feed consumption, body weight, and body weight gain during the dosing period followed by post-dosing recovery. There were no compound-related effects on any of the reproductive parameters examined or on foetal sex ratios or the incidence of gross, skeletal or visceral malformations in this study. Signs of foetal toxicity, decreased foetal body weight and an increased incidence of minor skeletal and visceral variations, were observed at the maternally toxic high-dose level. There was no evidence of embryotoxicity or teratogenicity in this study. The maternal and foetal no observable effect level in this study was estimated to be 50 mg/kg/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:

GLP compliant EPA 83-3

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 (Weissenborn 1987)

The foetotoxicity, teratogenicity and developmental toxicity of the substance to rats was studied under GLP to EPA 83-3. The test substance was administered by oral gavage at doses of 10, 50, or 250 mg/kg/day to three groups of rats (n=26) on gestational days 6-15, and a group of control animals (N=26) received an equivalent volume of the vehicle (10 mL/kg/day). No compound-related effects were observed in the low- or intermediate-dose groups. Signs of treatment related maternal toxicity observed in the high-dose group included salivation and reductions in feed consumption, body weight, and body weight gain during the dosing period followed by post-dosing recovery. There were no compound-related effects on any of the reproductive parameters examined or on foetal sex ratios or the incidence of gross, skeletal or visceral malformations in this study. Signs of foetal toxicity, decreased foetal body weight and an increased incidence of minor skeletal and visceral variations, were observed at the maternally toxic high-dose level. There was no evidence of embryotoxicity or teratogenicity in this study. The maternal and foetal no observable adverse effect level in this study was 50 mg/kg/day.

Developmental toxicity in rabbits (Wallace 1985)

The foetotoxicity, teratogenicity and developmental toxicity of the substance to rabbits was studied under GLP to OECD TG 414. The substance was administered by oral gavage at daily doses of 2, 12, and 72 mg/kg to New Zealand White rabbits (19 animals/group) from days 7 through 19 of gestation. Control animals received an appropriate volume of vehicle without test substance. Treatment-related effects of maternal toxicity were observed only at the high dose level, including reduction in feed consumption and in maternal body weight gain. In addition, vaginal bleeding was noticed. There were no treatment-related deaths in this study nor any adverse effects on any of the reproductive parameters examined, on foetal body weight, foetal sex ratios or on the incidence of foetal gross, visceral, or skeletal malformations. In two high-dose group foetuses a statistically significant increase in the incidence of incompletely ossified forepaw proximal phalanges was observed. This variation was considered to be attributable to the observed maternal toxicity and thought to represent a slight delay in normal development rather than direct foetotoxicity. The substance was not embryotoxic, foetotoxic, or teratogenic at doses as high as 72 mg/kg bw/day.

Justification for classification or non-classification

Based on the available information, classification for reproductive toxicity is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.