Thiophanate-methyl

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

In a two generation reproduction toxicity study similar to OECD TG 416 with one litter in the P and two litters in the F1 generation no adverse effects on fertility have been reported in rats.

The NOAEL for fertility and general reproductive performance is 147 mg/kg bw/day and 173 mg/kg bw/day for males and females.

The LOAEL for male and female parental animals in the P and F1 generation is 15 mg/kg bw/day and 18 mg/kg bw/day for males and females.

The NOAEL in the F1a is 55.4 mg/kg bw/day and 46 mg/kg bw/day for males and females.

The NOAEL in the F2b is 15 mg/kg bw/day and 18 mg/kg bw/day for males and females.

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 1991 - August 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

1983

Deviations:

yes

Remarks:

Weights of brain, kidneys and adrenal glands not recorded, Oestrus cycle, sperm parameters and number of implantations, corpora lutea and post-implantation loss not recorded

Qualifier:

according to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

Version / remarks:

1982

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD (SD)BR

Details on species / strain selection:

The Sprague Dawley rat was selected by the study sponsor as an appropriate species for the study because it is a readily available rodent species acceptable to regulatory authorities and because of the large historical control data base available for this strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, 97633 Sulzfeld, Germany
- Age at study initiation: (P) 6 wks; (F1) 5 wks
- Weight at study initiation: (P) Males:180 to 260 g; Females: 160 to 220 g; (F1) Males: 149-156 g; Females:138-145 g
- Fasting period before study: no
- Housing: Male and female rats were individually housed in solid floor macrolone cages with stainless steel lids except during mating, when one male was housed with one female and during lactation, when each female was housed with its litter.
- Diet: ad libitum, Ssniff R 10; Ssniff Spezialdiaten GmbH, 59494 Soest, Germany
- Water: ad libitum, tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: at 28-day intervals
- Mixing appropriate amounts with basic powdered diet (Ssniff R 10; Ssniff Spezialdiaten GmbH, 59494 Soest, Germany)
- Storage temperature of food: The formulation preparations were stored at room temperature.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: up to 21 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): housed individually.
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical results show that the test article concentrations in the formulations were close to the nominal concentrations (group 2: 81 to 114%; group 3: 89 to 120 %; group 4: 89 to 112 %of nominal concentration) as specified in the study protocol and that the test article was homogeneously admixed to the basic powdered diet. Stability testing covered intervals up to 35 days and revealed that during that interval no substantial test article loss occurred in the formulation preparations (group 2: 95 to 102 %; group 3: 96 to 105 %; group 4: 102 to 105 % of nominal concentration after 35 days at room temperature or deep frozen).

Duration of treatment / exposure:

The P animals were treated for 14 weeks and then allowed to mate for a maximum of 21 days to produce one litter (F1a).
Approximately 14 weeks after weaning of all F1a offspring, the selected F1 animals were allowed to mate for a maximum of 21 days within the same dosage group (avoiding sibling pairings) to produce one litter (F2a).
Because of unexpected mortality noted for F2a pups during the weaning period, a second mating was planned.
After weaning of the surviving F2a pups, the F1 animals were maintained for six weeks and then allowed to mate for a second time for a maximum of 21 days with the same partner as during the first mating, as applicable, to produce one further litter (F2b).

Frequency of treatment:

continously

Dose / conc.:

200 ppm (nominal)

Dose / conc.:

630 ppm (nominal)

Dose / conc.:

2 000 ppm (nominal)

No. of animals per sex per dose:

25

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
The dose levels were selected on the basis of the results of a dose range-finding study with Sprague Dawley male and female rats where dose levels of 75, 200, 1200 and 6000 ppm were used. Administration of 6000 ppm elicited toxic effects on the parental animals (i.e. markedly reduced body weight gain in male and female animals, markedly reduced food consumption in the male and slightly reduced food consumption in the female animals and markedly increased thyroid weights in males and females).
At 1200 ppm, also toxic effects were detected (i.e. slightly reduced body weight gain in the males and slightly to moderately increased thyroid weights in males and females). At 200 and 75 ppm, no adverse effects on male and female parental animals were elicited. The general reproductive performance of the P generation and development of the F1a offspring were not affected by treatment. On the basis of these results, dose levels of 200, 630 and 2000 ppm were selected for the main study.

Positive control:

none

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked: morbidity and mortality

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of parental male and female animals was recorded once weekly during the premating and the mating periods. The body weight of the female animals was additionally recorded on days 0, 7, 14 and 20 post-coitum. During lactation, all dams were weighed on days 1, 4, 7, 14 and 21 post-partum. Additionally, body weight of all animals was recorded at terminal kill.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Food consumption of parental males and females was recorded twice weekly (intervals of 3 days and 4 days) during the premating period. Food consumption data were evaluated for weekly intervals.
Additionally, food consumption of the females was recorded on days 0, 3, 7, 10, 14, 17 and 20 post-coitum and the data were evaluated for the interval from day 0 to 7, 7 to 14 and 14 to 20 post-coitum.
During lactation, food consumption of the females was recorded on days 0, 4, 7, 9, 11 and 13 and then daily until day 21 post-partum. Evaluation of the data was performed for the intervals from days 0 to 4, 4 to 7, 7 to 14 and 14 to 21 post-partum.
Food consumption was calculated as mean daily food consumption per measuring period.

WATER CONSUMPTION AND COMPOUND INTAKE: No

OTHER:
Blood sampling was performed on ten male and ten female animals per group during the first week of treatment, in week 8 of treatment and at necropsy. Blood samples were taken from identical animals at each occasion, if possible.
The serum was divided into two sub-samples per animal and stored deepfrozen (if not analysed on the same day) and submitted for thyroid hormone determination as follows:
- T3 and T4 were determined by luminescense immunoassay (LIA) in HAZLET0N DEUTSCHLAND's analytical laboratory.
- TSH was determined by radio immunoassay (RIA) at HAZLET0N WASHINGTON.

Oestrous cyclicity (parental animals):

not determined

Sperm parameters (parental animals):

Parameters examined in [P/F1] male parental generations:
testis weight, epididymis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 ] offspring:
number and sex of pups, live births, postnatal mortality, presence of gross anomalies, individually weighed on days 1, 4, 7, 14 and 21 post-partum

During lactation, the pups in each litter were examined daily and the physical development of the progeny was assessed by monitoring the following events:
Pinna unfolding: the day on which the pinnae became detached;
Tooth eruption: the day on which the upper incisors were observed to penetrate the gum;
Eye opening: the day on which the upper and lower eyelids separated.
For each developmental event, the number of pups in each litter showing the observation was daily recorded until all the pups in the litter showed the observation.

For each pup, the following functional tests were performed:
- surface righting reflex, examined on day 8 post-partum;
- gripping reflex, examined on day 17 post-partum;
- pupillary reflex, examined on day 21 post-partum;
- auditory response, examined on day 21 post-partum.

Additionally, those F1a pups, which were selected for the F1 generation were examined for the following parameter:
- open field test, performed at 5 weeks of age.

GROSS EXAMINATION OF DEAD PUPS:
Pups dying or killed during lactation were examined for external and visceral abnormalities.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: Yes

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

Postmortem examinations (parental animals):

SACRIFICE
All parental male and female animals of the P generation were killed by carbon dioxide inhalation and necropsied after weaning of the Fla progeny.
All Fl parental male and female animals were killed by carbon dioxide inhalation and necropsied after weaning of the F2b offspring.
Non-selected F1a weanlings were killed after selection of the F1 generation. All F2a and F2b weanlings were killed and necropsied shortly after weaning.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY
The following tissues of all parental animals of the P and F1 generation - if applicable - were preserved in 10 per cent neutral buffered formalin (except testes and epididymides which were fixed in Bouin's fluid):
- cervix
- coagulating gland
- epididymides*
- liver*
- ovaries
- pituitary
- prostate
- seminal vesicles
- testes*
- thyroids*
- uterus
- vagina
- organs which macroscopically showed alterations

All organs marked with "*" were weighed before fixation, paired organs were weighed separately.
The tissues of the control and high dose group animals were embedded in paraffin wax, sectioned at a nominal thickness of 5 µm stained with hematoxylin and eosin, and examined by the study histopathologists.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed shortly after weaning.
- These animals were subjected to postmortem examinations (macroscopic examination)

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

Statistics:

Different statistical methods for the evaluation of the different parameters (e. g. Levene's test, ANOVA, ANCOVA, Dunnett's test, Bartlett's test, Kruskal-Wallis test).

Reproductive indices:

see " Any other information on material and methods"

Offspring viability indices:

see " Any other information on material and methods"

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related findings were detected in the male and female animals of this generation.
Minor clinical observations such as hair loss and rough haircoat were noted in single male animals of the control group and of group 4 (2000 ppm). Single clinical observations such as hair loss, minor injuries, or vaginal discharge were sporadically noted in female animals.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

A total of four mortalities was observed in the female animals of this generation; one from the control group, two from the low dose and one from the intermediate dose group. None of these mortalities in the dose groups is considered to be due to treatment. No mortalities were observed in the male animals of the P generation.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the male animals of this generation, mean body weight change was reduced (9.6 % lower than the control) in group 4 (2000 ppm) during the premating and mating period. This finding is considered related to treatment. Although minor differences (increases and decreases) were noted in weekly body weight change data, overall body weight change of the female animals of the high dose group was consistent with that of the control group.
During gestation and lactation, expected variations of mean body weight change were observed but no treatment-related effects were noted.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

In males and females of the P generation, mean daily food consumption was not affected by treatment.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

T3, T4 and TSH were the thyroid hormonal levels analysed in ten female and ten male animals of the P generation in weeks 1 and 8 and prior to sacrifice (beginning in week 32). Possible treatment-related effects on T4 and TSH were noted for the male animals of group 4 (2000 ppm).

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related histopathological findings were detected in liver and thyroid of male and female animals of the high dose group. Thyroid findings were more frequent in the male animals.
In 22 out of 25 male animals of group 4 (2000 ppm), centrilobular hepatocyte hypertrophy in the liver was found. None of the males in the control group showed this finding. This finding is considered to represent a functional adaptive change to an increased metabolism in response to a foreign compound. Therefore, this finding is considered attributable to treatment.
Follicular cell hypertrophy and hyperplasia were detected in the thyroid of 22 and 21 of the 25 males of the high dose group, respectively (versus 0 respective 1 in the control group). These findings are consistent with prolonged stimulation of the thyroid gland, reflect the findings in the thyroid hormonal levels found, and represent an effect of treatment.
In the female animals of the high dose group, hepatocyte hypertrophy was found in 18 of the 25 examined females of this dose group (versus 0 in the control group) and follicular cell hypertrophy and hyperplasia in the thyroid in 6 and 5 females, respectively (versus none in the control group). These findings are considered to be treatment-related.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Description (incidence and severity):

Sperm counts were not investigated; however there were no histopathological effects on testis.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Mating performance, indicated as duration (in days) of cohabitation until day 0 of gestation, showed the usual inter-group variations but no treatment-related effect.
In group 4 (2000 ppm), mating performance was comparable to that of the control group. The slightly higher mean value noted for the high dose group was due to one mating pair; 14 cohabitation days for female no. 80 and male no. 180. Exclusion of this value from calculation of mating performance leads to identical results (2.0 days in group 4 and in the control group).
The other reproduction indices as insemination, fecundity and fertility index also showed the usual slight variations between the single groups but they were of no treatment-related relevance.

Key result

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: highest dose tested

Key result

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: highest dose tested

Key result

Dose descriptor:

NOAEL

Remarks:

parental toxicity

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Remarks:

parental toxicity

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

During the examination periods prior to and during the first as well as the second mating, minor clinical observations as bloody crust around eyes and rough haircoat were noted in one or more animals of the F1 generation but none of them with any relation to treatment. In the female animals of this generation, single clinical observations such as hair loss, bloody crust around eyes, or vaginal discharge were sporadically detected during the examination periods prior to, during, and after the first and second mating. Nature and incidence of these findings did not indicate any treatment-related effect.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two mortalities were seen in the male animals of this generation; one from the intermediate (animal no. 357) and one from the high dose (animal no. 375). At necropsy, no findings were detected which could be related to treatment. These deaths are considered sporadic occurrences. A single death occurred in the female animals of this generation prior to the first mating: one low dose animal died (animal no. 228). On the day prior to its death, this animal had shown clinical signs as pale and cool to touch. Necropsy of this animal did not reveal any treatment-related findings. Therefore, this mortality is considered incidental and not treatment-related.
Prior to termination of the study after second mating, three further females died: female no. 204 from the control group died on day 69 showing vaginal discharge and incomplete delivery. Animal no. 237 from the 200 ppm group was also found dead on day 69. This aiimal showed vaginal discharge and was pale and cool to touch. Female no. 261 from group 3 (630 ppm) was found dead on day 84 after having been hunched and pale the day before.
Necropsy of these females which died prior term revealed signs of incomplete delivery in female nos. 204 and 261. In animal no. 237, no remarkable observations were made at necropsy. These mortalities are considered incidental .

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the male animals of this generation, mean body weight change was reduced (5.6 % lower than control) in group 4 (2000 ppm) prior to and during the first and second mating. Females receiving 2000 ppm showed reduced body weight change during the first premating period (7.4 % lower than control) and during first gestation (11.9 % lower than control). These findings are considered attributable to treatment.
In the male animals of group 3 (630 ppm), overall body weight gain prior to and during the first mating period was lower (3.1 %) than control. Body weight changes prior and during to the second mating were statistically significantly lower than control. These differences in body weight gain are considered treatment-related.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Mean daily food consumption of the male animals prior to the first and second mating was generally similar to the control group for all treated groups.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In male and female animals of the high dose group in the F1 generation, TSH values were statistically significantly higher in week 8. In the female animals, TSH values were also numerically higher at necropsy. These findings are considered treatment-related. In this dose group, T3 and T4 values did not indicate any treatment-related effect.
In the male animals of the intermediate dose group, the TSH value was also elevated in week 8. This is considered equivocal. The other hormonal parameters examined for the males of this dose group were comparable with the control.
The F1 animals of the low dose group did not show any treatment-related effects on the hormonal levels examined.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The functional tests performed on days 8 post-partum (i.e. surface righting reflex), 17 post-partum (gripping reflex), 21 post-partum (pupillary reflex and auditory response) and 35 to 37 post-partum (open field test) did not reveal any effects of treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The liver and thyroid weights for the male animals of the high dose group were statistically significantly higher than in the control group. As organ weight results from the P generation also revealed statistically significantly higher thyroid and liver weights from the high dose males, these increased weights for the thyroid and liver of the F1 high dose males are considered treatment-related. The liver weight of the males in group 2 (200 ppm) was also higher than the control and the difference to the control was also statistically significant but with regard to the liver weight determined in the intermediate dose group and for the P generation males of the low dose group, this finding is considered not to be attributable to treatment. Thyroid and liver weights found in group 3 (630 ppm) and thyroid weight found in the low dose group (200 ppm) were generally comparable with the control.
As noted for the males, the liver and thyroid weights for the female animals of the high dose group of this generation were higher than in the control (thyroid weight statistically significantly higher and liver weight numerically higher). As similar findings (statistically significant) were noted for the high dose females from the P generation, the increased liver and thyroid weights for the high dose (2000 ppm) F1 females are considered treatment-related.
Thyroid weights from the low and intermediate dose groups were also statistically significantly higher than in the control group but the differences were clearly smaller than between high dose and control group. Therefore, and with regard to the thyroid weights observed in these dose groups in the P generation, these findings are considered not to be treatment-related.
Liver weights of the F1 females in the low and intermediate dose group were comparable with the control.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In the two male animals which died before term, one each from the intermediate and high dose group, necropsy findings in intestines (such as dark red contents), kidneys (hydronephrosis) and urinary bladder (uroliths, dark red mucosa) were detected. None of these findings was related to treatment. In the male animals necropsied at term, the usual sporadic findings were detected in the abdominal organs (such as renal pelvis dilatation, testis reduced in size). These findings did not indicate any treatment-related effect.
In the female animal from group 2 (200 ppm) which died before term during the first mating period, necropsy revealed findings in the thoracic cavity (filled with watery fluid) and abdominal organs (liver pale with red spots, uterine horn filled with placental remnants and red fluid). These findings are considered not to be attributable to treatment.
In the animals which died before term during the second mating period, signs of incomplete delivery were detected (in female nos. 204 and 261) but no findings which are related to treatment.
In the other females, some single findings were detected in the abdominal organs (such as kidneys mottled, swelling or wound in the skin). These findings were of minor nature and/or low incidence and are therefore considered not to be attributable to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The only treatment-related histopathological findings were detected in liver of male and female animals of the high dose group and thyroid of the male animals of the high dose group.

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):

Best mating performance, i.e. the lowest mean number of cohabitation days until day 0 of gestation was observed in group 4 (2000 ppm). In the other dose groups, mating performance was only minimally different from that of the control group and these differences are considered to be incidental. The insemination indices of the dose groups were similar to that of the control group.
In 6 out of 24 male animals of group 4 (2000 ppm), centrilobular hepatocyte hypertrophy in the liver was found. None of the males in the control group showed this finding. This finding is considered to represent a functional adaptive change to an increased metabolism in response to a foreign compound. Therefore, this finding is considered attributable to treatment. Follicular cell hypertrophy and hyperplasia were detected in the thyroid of 6 respective 20 of the 24 males of the high dose group versus none in the control group. These findings are considered signs of prolonged stimulation of the thyroid gland and to represent an effect of treatment.
In the female animals of the high dose group, the same liver findings were detected: hepatocyte hypertrophy was found in 5 of the 25 examined females of this dose group versus 0 in the control group. This finding is considered to be treatment-related. In the thyroids of the female animals of this generation, treatment-related findings were not seen.

Mating performance of the F1 animals during the second mating, i.e. the mean number of cohabitation days until day 0 of gestation, was comparable in all groups and did not indicate any treatment-related effect.

Key result

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: highest dose tested

Key result

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: highest dose tested

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

Key result

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The mean number of live pups on day 1 post-partum was similar in all groups. Accordingly, live birth indices did not reveal any inter-group differences. During lactation, pup losses were observed in all groups and they were comparable for all examination intervals. Therefore, viability and weaning indices were similar in all groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean F1a pup weight in group 4 (2000 ppm) was comparable with the control until day 4 post-partum. On days 7, 14 and 21 post-partum, lower mean pup weights (male and female) in comparison with the control group were observed in this dose group. Although these differences did not achieve statistical significance, these lower mean pup weights are considered to be related to treatment.
In groups 2 (200 ppm) and 3 (630 ppm), mean pup weights were minimally reduced.

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

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Necropsy of the F1a pups did not reveal treatment-related findings.

Histopathological findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The functional tests performed on days 8 post-partum (i.e. surface righting reflex), 17 post-partum (gripping reflex), 21 post-partum (pupillary reflex and auditory response) and 35 to 37 post-partum (open field test) did not reveal any effects of treatment.

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1a

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

Key result

Dose descriptor:

NOAEL

Generation:

F1a

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

Key result

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

F2a: Unexpected, very high pup losses were observed for all groups. The majority of the deaths occurred after day 4 of lactation. The weaning indices (number alive at weaning / number alive at day 4 postcull) for the control, low, intermediate and high dose group were 12 %, 24 %, 13 % and 13 %, respectively. Evaluation of all relevant data did not reveal a clear explanation for the pup losses. Evaluation of the data of the surviving pups showed moderately reduced mean weight in the male pups, of group 4 (2000 ppm) as the only treatment-related effect. It is therefore concluded that those pups which did not die showed a normal and unaffected post-natal development (except in the high dose group where the male pups showed reduced body weight gain).
F2b: Results of the second breeding were consistent with those noted previously for studies for which a second mating was required. The general trend is for fewer litters to be produced as a reflection of the age of the parental animals. The number of females with liveborn pups were 17, 19, 14 and 15 for the control , 200 ppm, 630 ppm and 2000 ppm groups, respectively. These values are comparable with historical control data.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

F2a: Evaluation of the data of the surviving pups showed moderately reduced mean weight in the male pups, of group 4 (2000 ppm) as the only treatment-related effect. It is therefore concluded that those pups which did not die showed a normal and unaffected post-natal development (except in the high dose group where the male pups showed reduced body weight gain).
F2b: Body weights were comparable to the control values except for males at the highest dose at 21 days post-partum for which mean body weigh was moderately lower than in the control group. This was considered by the study author to be treatment related. In F2b pups, reduced body weight gain was noted from 630 ppm onward, with a slight (-9%) but statistically significant reduction also at 14 days at 200 ppm.

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):

F2a: The sex distribution of the F2a pups on days 1 and 21 post-partum was comparable in all groups. This indicates that the observed high pup losses were not sex related.
F2b: The sex distribution of the F2b pups on days 1, 4 precull and 21 post-partum was comparable in all groups and not affected by treatment.

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

F2a: Necropsy of F2a pups revealed the usual findings in stomach (without contents) and spleen (enlarged) but did not reveal any treatment-related effects.
F2b: Necropsy of pups revealed the usual findings e.g. in stomach (without contents), liver (prominent lobular pattern, pale), kidneys (enlarged, renal pelvis dilatation) and urinary bladder (enlarged, floccules) but did not reveal any treatment-related effects.

Histopathological findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

F2a: The physical parameters examined as pinna unfolding, incisor eruption and eye opening did not reveal any treatment-related differences between the dose groups and the control group. The functional tests performed in the F2a pups as surface righting, gripping, pupillary and auditory reflexes on day 21 post-partum did not indicate any effect of treatment.
F2b: The physical parameters examined as pinna unfolding, incisor eruption and eye opening did not reveal any treatment-related differences between the dose groups and the control group. The functional tests performed in the F2b pups as surface righting, gripping, pupillary and auditory reflexes did not indicate any effect of treatment.

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F2b

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

Key result

Dose descriptor:

NOAEL

Generation:

F2b

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Table 1: Test substance intake

Dose levels (ppm)	200	630	2000
Premating period males	14.6	46.0	147.1
Premating period females	16.8	52.2	164.3
Gestation period females	14.3	44.6	138.2
Lactation period females	27 2	81.2	247.1
Overall females	18.0	55.4	172.9

 

Table 2: F1a pup weights, covariate adjusted means (covariate: number of pups)

Dose levels (ppm)	0		200		630		2000
	m	f	m	f	m	f	m	f
day 14	28.96	28.46	27.99	27.29	27.03	27.27	26.20 (-10%)	25.29 (-11%)
day 21	49.26	47.38	48.05	46.63	45.68	46.13	44.62 (-9%)	42.66 (-10%)

 

Table 3: F2a pup weights, covariate adjusted means (covariate: number of pups)

Dose levels (ppm)	0		200		630		2000
	m	f	m	f	m	f	m	f
day 14	29.34	28.74	28.14	23.88 (-17%)	23.23	22.08	25.47 (-13%)	25.02 (-13%)
day 21	50.12	46.10	44.77 (-11%)	38.24 (-17%)	51.21	43.57	46.76 (-7%)	48.44 (-5%)

Table 4: F2b pup weights, covariate adjusted means (covariate: number of pups)

Dose levels (ppm)	0		200		630		2000
	m	f	m	f	m	f	m	f
day 14	33.09	31.38	30.27* (-9%)	30.22 (-4%)	29.79* (-10%)	27.23* (-15%)	28.84** (-13%)	26.46** (-16%)
day 21	54.73	51.69	52.10 (-5%)	51.23 (-1%)	49.53 (10%)	45.32* (-12%)	48.30* (-12%)	45.30* (-12%)

* Statistically significantly different from control. p<0.05

* * Statistically significantly different from control. p<0.01

 Table 5: Histopathological findings and organ weights of liver and thyroid of P and F1 generation rats

Sex	Males				Females
Dose (ppm)	0	200	630	2000	0	200	630	2000
P-generation
Hepatocyte hypertrophy Study pathologist	0/25	9/25	14/25	23/25	0/24	1/23	3/24	20/25
Peer reviewer	0/25	7/25	13/25	24 25	0/25	0/25	2/24	23 25
Liver weight (mg)	24433	24993	24731	28S36*	12053	12117	12142	13640*
Thyroid hyperplasia Study pathologist	0/25	6/25	13/24	17/25	0/24	0/23	2/24	5/25
Peer reviewer	0/25	4/25	11/24	16/25	0/25	0/24	1/25	7/25
Thyroid hypertrophy Study pathologist	0/25	9/25	7/24	22/25	0/24	0/23	1/24	4/25
Thyroid weight (mg)	29	31	29	39*	23	22	33	30*
F1-generation
Hepatocyte hypertrophy Study pathologist	0/25	3/25	6/24	11/24	0/24	1/23	1/24	15/25
Peer reviewer	0/25	1/25	7/25	15/25	0/25	0/24	1/25	18/25
Liver weight (mg)	20638	25289*	22956	26015*	13341	13651	13445	14274
Thyroid hyperplasia Study pathologist	0/25	4/25	4/24	10/24	3/24	0/23	1/24	2/22
Peer reviewer	0/25	1/25	3/25	9/24	3/24	0/24	1/25	9/22
Thyroid hypertrophy Study pathologist	0/25	3/25	2/24	1/24	3/24	0/23	0/24	0/22
Thyroid weight (mg)	28	31	30	36*	23	26*	26*	29*

*: p<0.05; Statistical significance of histopathological findings not given.

 

Conclusions:

In conclusion, this two generation reproduction toxicity study with one litter in the P and two litters in the F1 generation where the substance was administered at 200 ppm, 630 ppm, and 2000 ppm revealed the following results:
The NOAEL for fertility and general reproductive performance is 147 mg/kg bw/day and 164 mg/kg bw/day for males and females.
The NOAEL for male and female parental animals in the P and F1 generation is 15 mg/kg bw/day and 18 mg/kg bw/day for males and females.
The NOAEL in the F1a is 55.4 mg/kg bw/day and 46 mg/kg bw/day for males and females.
The NOAEL in the F2b is 15 mg/kg bw/day and 18 mg/kg bw/day for males and females.

Executive summary:

A study similar to OECD TG 416 was performed and the test item was administered via the diet to approximately 6 weeks old Sprague-Dawley Crl:CD (SD)BR rats. For the parental generation (P) 25 males and females per dose group were used. The doses were 0, 200, 630 and 2000 ppm. The selection of doses based on a range-finding study with doses of 0, 75, 200, 1200 and 6000 ppm. After 14 weeks of premating treatment, the P animals were mated for up to 21 days. The P females were allowed to litter and to rear their offspring (F1a generation) to weaning. Following a 14 weeks maturation period after weaning, the F1 parental animals (selected from the F1a offspring) were mated for up to 21 days. The F1 females were allowed to litter and to rear their offspring (F2a generation) to weaning. Because of an unusually high pup loss during the weaning period of the F2a pups a second mating was performed. The F1 animals were maintained on test substance/diet for six weeks and then allowed to mate with the same partner as during the first mating to produce an F2b-litter.

All animals were examined at least daily for signs of clinical toxicity. Body weight of parental male and female animals was recorded once weekly during the premating and mating periods. The body weight of the females was additionally recorded on days 0, 7, 14 and 20 post-coitum. During lactation, all dams were weighed on days 1, 4, 7, 14 and 21 post-partum. Food consumption was recorded twice weekly during the premating periods, additional determinations were made during gestation and lactation. Blood sampling was performed on 10 animals per sex from the P and F1 generations. The serum was used for the determination of thyroid hormone (T3 and T4) and thyroid stimulating hormone (TSH) concentrations.

For each pregnant female the date of mating, date of parturition, duration of gestation, abnormalities of nesting and nursing behaviour and the number of implantations were recorded. The size of each litter was adjusted to four male and four female pups on day 4 post-partum by random selection. During lactation, the pups in each litter were examined daily and their development was monitored by pinna unfolding, tooth eruption and eye opening. For each pup the following functional tests were performed:

- surface righting reflex (day 8 post-partum)

- gripping reflex (day 17 post-partum)

- pupillary reflex (day 21 post-partum)

- auditory response (day 21 post-partum)

- open field test (F1a pups, selected for the F1 parental generation, at 5 weeks of age).

All parental male and female animals of the P generation were sacrificed after weaning of the F1a progeny. All F1 parental male and female animals were sacrificed after weaning of the F2b offspring. Non-selected F1a weanlings were sacrificed after the selection of the F1 generation. All F2a and F2b weanlings were sacrificed shortly after weaning. All animals were necropsied. Histopathological examination was carried out according to the guideline with special reference to the reproduction organs. Because of treatment-related histopathological effects in the liver and thyroid at the high dose, these organs from all P and F1 animals at 200 and 630 ppm were further evaluated.

Parental generation

There were no treatment-related deaths or clinical findings in any of the parental males and females.

Mean daily food consumption was only affected in the F1 females of the high dose group (prior to first mating, during the first and second gestation and during the first lactation period). Reduced body weight gain was observed at 2000 ppm in the male animals of the P generation (-10%) as well as in males and females of the Fl generation (males: ca. -6%; females: ca. -7% during the premating period and ca. -12% during gestation). In the males of the intermediate group, the overall body weight gain was slightly but significantly lower than that of the control group at a series of time points.

No treatment-related effects were noted on mating performance, insemination, fecundity or fertility index. No compound-related clinical signs of impaired pregnancy or delivery were observed.

Litter generation             

Findings at and post birth

No treatment-related effects were noted on gestation time, live birth index, viability index, weaning index or sex distribution.

Findings during lactation period

Unusually high pup losses were observed in the F2a generation for all groups after day 4 of lactation. The weaning indices (number alive at weaning/number alive at day 4 postcull) for the control, low, intermediate and high dose groups were 12%, 24%, 12% and 13%, respectively. Thorough evaluation of all relevant parameters (animal room conditions; diet, test article and water supply, condition of cages, food hoppers and bedding material, time relations of deaths, etc.) did not reveal any reason for these pup losses, but led to the conclusion that this finding was a random event due to biological variation. Similar findings were occasionally seen in the performing laboratory before. The pups alive showed a normal postnatal development (except the reduced body weight gain in the high dose males). The weaning indices for the F2b groups were 94%, 74%, 79% and 87%.

Mean pup weight was reduced in both generations at the highest dose on days 14 and 21 but not at earlier time points. In F1a the differences (up to 11% reduction) did not achieve statistical significance but were considered to be related to treatment. In F2a pups, body weights were comparable to the control values except for males at the highest dose at 21 days post-partum for which mean body weight was moderately lower than in the control group. This was considered to be treatment related. In F2b pups, reduced body weight gain was noted from 630 ppm onward, with a slight (-9%) but statistically significant reduction also at 14 days at 200 ppm.

Findings after weaning

There were no substance related necropsy findings in the F2a and F2b pups. The functional tests performed, i.e. surface righting reflex, gripping reflex, pupillary reflex and auditory response and open field test, did not reveal any treatment-related findings. In male pups of the 2000 ppm dose group, the mean number of entered squares was lower compared to the control group. Due to the high standard deviations and lack of a similar finding in the female animals of this group, this finding was considered to be incidental.

Necropsy findings, organ weights and thyroid hormones:

In the 2000 ppm group, the males and females of the P and F1 generations showed elevated TSH levels at the 8 week measuring point. In the males of this dose group from the P generation, lower T4 values were also considered attributable to treatment. In high dose P females, reduced T4 values were noted only in the first week. Effects on T3 values were marginal or non-existent. The occasional numerical changes in hormone values in the 630 ppm groups were equivocal. The changes in hormonal parameters in the high dose group were consistent with the histopathological findings and changed organ weights. In this dose group, thyroid and liver weights were increased in male and female animals of both generations. Because of the findings at the high dose the liver and thyroid from the low and mid dose parent animals (P and F1 generations) were also evaluated. Further, a peer review referring to the histopathological findings and the organ weight analysis was performed. The incidences of hypertrophy in the thyroid were not extra listed in the table by the peer reviewer, but as he pointed out, the hyperplasia was generally accompanied by hypertrophy. Combining the results of both, the histopathological and organ weight data and including the fact that the F1 generation should be more vulnerable than the parent generation because of the exposure in utero, during nursing and during growth, the dose of 200 ppm was concluded by the Peer reviewer to be at most an equivocal effect level.

In conclusion, this two generation reproduction toxicity study with one litter in the P and two litters in the F1 generation where the substance was administered at 200 ppm, 630 ppm, and 2000 ppm revealed the following results:

The NOAEL for fertility and general reproductive performance is 147 mg/kg bw/day and 164 mg/kg bw/day for males and females.

The NOAEL for male and female parental animals in the P and F1 generation is 15 mg/kg bw/day and 18 mg/kg bw/day for males and females.

The NOAEL in the F1a is 55.4 mg/kg bw/day and 46 mg/kg bw/day for males and females.

The NOAEL in the F2b is 15 mg/kg bw/day and 18 mg/kg bw/day for males and females.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

147 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

similar to OECD TG 416

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A study similar to OECD TG 416 was performed and the test item was administered via the diet to approximately 6 weeks old Sprague-Dawley Crl:CD (SD)BR rats. For the parental generation (P) 25 males and females per dose group were used. The doses were 0, 200, 630 and 2000 ppm. The selection of doses based on a range-finding study with doses of 0, 75, 200, 1200 and 6000 ppm. After 14 weeks of premating treatment, the P animals were mated for up to 21 days. The P females were allowed to litter and to rear their offspring (F1a generation) to weaning. Following a 14 weeks maturation period after weaning, the F1 parental animals (selected from the F1a offspring) were mated for up to 21 days. The F1 females were allowed to litter and to rear their offspring (F2a generation) to weaning. Because of an unusually high pup loss during the weaning period of the F2a pups a second mating was performed. The F1 animals were maintained on test substance/diet for six weeks and then allowed to mate with the same partner as during the first mating to produce an F2b-litter.

All animals were examined at least daily for signs of clinical toxicity. Body weight of parental male and female animals was recorded once weekly during the premating and mating periods. The body weight of the females was additionally recorded on days 0, 7, 14 and 20 post-coitum. During lactation, all dams were weighed on days 1, 4, 7, 14 and 21 post-partum. Food consumption was recorded twice weekly during the premating periods, additional determinations were made during gestation and lactation. Blood sampling was performed on 10 animals per sex from the P and F1 generations. The serum was used for the determination of thyroid hormone (T3 and T4) and thyroid stimulating hormone (TSH) concentrations.

For each pregnant female the date of mating, date of parturition, duration of gestation, abnormalities of nesting and nursing behaviour and the number of implantations were recorded. The size of each litter was adjusted to four male and four female pups on day 4 post-partum by random selection. During lactation, the pups in each litter were examined daily and their development was monitored by pinna unfolding, tooth eruption and eye opening. For each pup the following functional tests were performed:

- surface righting reflex (day 8 post-partum)

- gripping reflex (day 17 post-partum)

- pupillary reflex (day 21 post-partum)

- auditory response (day 21 post-partum)

- open field test (F1a pups, selected for the F1 parental generation, at 5 weeks of age).

All parental male and female animals of the P generation were sacrificed after weaning of the F1a progeny. All F1 parental male and female animals were sacrificed after weaning of the F2b offspring. Non-selected F1a weanlings were sacrificed after the selection of the F1 generation. All F2a and F2b weanlings were sacrificed shortly after weaning. All animals were necropsied. Histopathological examination was carried out according to the guideline with special reference to the reproduction organs. Because of treatment-related histopathological effects in the liver and thyroid at the high dose, these organs from all P and F1 animals at 200 and 630 ppm were further evaluated.

Parental generation

There were no treatment-related deaths or clinical findings in any of the parental males and females.

Mean daily food consumption was only affected in the F1 females of the high dose group (prior to first mating, during the first and second gestation and during the first lactation period). Reduced body weight gain was observed at 2000 ppm in the male animals of the P generation (-10%) as well as in males and females of the Fl generation (males: ca. -6%; females: ca. -7% during the premating period and ca. -12% during gestation). In the males of the intermediate group, the overall body weight gain was slightly but significantly lower than that of the control group at a series of time points.

No treatment-related effects were noted on mating performance, insemination, fecundity or fertility index. No compound-related clinical signs of impaired pregnancy or delivery were observed.

Litter generation             

Findings at and post birth

No treatment-related effects were noted on gestation time, live birth index, viability index, weaning index or sex distribution.

Findings during lactation period

Unusually high pup losses were observed in the F2a generation for all groups after day 4 of lactation. The weaning indices (number alive at weaning/number alive at day 4 postcull) for the control, low, intermediate and high dose groups were 12%, 24%, 12% and 13%, respectively. Thorough evaluation of all relevant parameters (animal room conditions; diet, test article and water supply, condition of cages, food hoppers and bedding material, time relations of deaths, etc.) did not reveal any reason for these pup losses, but led to the conclusion that this finding was a random event due to biological variation. Similar findings were occasionally seen in the performing laboratory before. The pups alive showed a normal postnatal development (except the reduced body weight gain in the high dose males). The weaning indices for the F2b groups were 94%, 74%, 79% and 87%.

Mean pup weight was reduced in both generations at the highest dose on days 14 and 21 but not at earlier time points. In F1a the differences (up to 11% reduction) did not achieve statistical significance but were considered to be related to treatment. In F2a pups, body weights were comparable to the control values except for males at the highest dose at 21 days post-partum for which mean body weight was moderately lower than in the control group. This was considered to be treatment related. In F2b pups, reduced body weight gain was noted from 630 ppm onward, with a slight (-9%) but statistically significant reduction also at 14 days at 200 ppm.

Findings after weaning

There were no substance related necropsy findings in the F2a and F2b pups. The functional tests performed, i.e. surface righting reflex, gripping reflex, pupillary reflex and auditory response and open field test, did not reveal any treatment-related findings. In male pups of the 2000 ppm dose group, the mean number of entered squares was lower compared to the control group. Due to the high standard deviations and lack of a similar finding in the female animals of this group, this finding was considered to be incidental.

Necropsy findings, organ weights and thyroid hormones:

In the 2000 ppm group, the males and females of the P and F1 generations showed elevated TSH levels at the 8 week measuring point. In the males of this dose group from the P generation, lower T4 values were also considered attributable to treatment. In high dose P females, reduced T4 values were noted only in the first week. Effects on T3 values were marginal or non-existent. The occasional numerical changes in hormone values in the 630 ppm groups were equivocal. The changes in hormonal parameters in the high dose group were consistent with the histopathological findings and changed organ weights. In this dose group, thyroid and liver weights were increased in male and female animals of both generations. Because of the findings at the high dose the liver and thyroid from the low and mid dose parent animals (P and F1 generations) were also evaluated. Further, a peer review referring to the histopathological findings and the organ weight analysis was performed. The incidences of hypertrophy in the thyroid were not extra listed in the table by the peer reviewer, but as he pointed out, the hyperplasia was generally accompanied by hypertrophy. Combining the results of both, the histopathological and organ weight data and including the fact that the F1 generation should be more vulnerable than the parent generation because of the exposure in utero, during nursing and during growth, the dose of 200 ppm was concluded by the Peer reviewer to be at most an equivocal effect level.

In conclusion, this two generation reproduction toxicity study with one litter in the P and two litters in the F1 generation where the substance was administered at 200 ppm, 630 ppm, and 2000 ppm revealed the following results:

The NOAEL for fertility and general reproductive performance is 147 mg/kg bw/day and 173 mg/kg bw/day for males and females.

The LOAEL for male and female parental animals in the P and F1 generation is 15 mg/kg bw/day and 18 mg/kg bw/day for males and females.

The NOAEL in the F1a is 55.4 mg/kg bw/day and 46 mg/kg bw/day for males and females.

The NOAEL in the F2b is 15 mg/kg bw/day and 18 mg/kg bw/day for males and females.

Effects on developmental toxicity

Description of key information

In a study according OECD TG 414 in rats the NOAEL for developmental toxicity was 1000 mg/kg bw/day, the highest dose. NOAEL for maternal toxicity was 300 mg/kg bw/day based on reductions in body weight gain at 1000 mg/kg bw/day.

In a study according to OECD TG 414 in rabbits the NOAEL for maternal toxicity was 10 mg/kg bw/day based on reductions in maternal body weight gain and absolute and relative feed consumption. The developmental NOAEL was set at 20 mg/kg bw/day based on supernumerary thoracic ribs at 40 mg/kg bw/day.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 1997 - November 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

1981

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Covance Research Products, Inc, Denver, Pennsylvania, USA
- Age at study initiation: 6 month
- Weight at study initiation: 3.27-4.51 kg
- Fasting period before study: no
- Housing: individually
- Diet: From the day of arrival until DG 6, each rabbit was given approximately 150 g of Certified Rabbit Chow® #5322 (Purina Mills, Inc.) daily in an individual stainless-steel "J-type" feeder attached to each cage. Beginning on DG 6, the first day of dosage, each rabbit was given approximately 180 g of Certified Rabbit Chow®#5322 daily.
- Water: ad libitum, local water that had been processed by passage through a reverse osmosis membrane
- Acclimation period: one day

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16-22
- Humidity (%): 30-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 1% aqueous methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations of the test substance were prepared weekly. The test substance was considered 100% pure for the purpose of dosage calculations.

VEHICLE
- Justification for use and choice of vehicle: Substance no-soluble in water.
- Amount of vehicle: 10 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration and homogeneity analyses indicated no test substance contamination of the vehicle, all samples for the 0.5, 1.0, 2.0 and 4.0 mg/mL concentrations were within ±10% of target (5, 10, 20 and 40 mg/kg/day dosages, respectively) and the formulations were homogeneous. Stability analyses indicated the formulations were stable for a period of at least 7 days when stored refrigerated.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant

Duration of treatment / exposure:

gestation day 6 to 28

Frequency of treatment:

daily

Dose / conc.:

5 mg/kg bw/day (nominal)

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

20 mg/kg bw/day (nominal)

Dose / conc.:

40 mg/kg bw/day (nominal)

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
In a pilot study using two routes of oral exposure (stomach tube or diet) were selected to allow comparison of exposure. Six pregnant rabbits per test group were exposed to 0, 5, 10, 20, 40 and 80 mg/kg/day from GD 6 to 18. Based on data of this preliminary study, dosages of 80 mg/kg bw/day of thiophanate-methyl either in the diet or via gavage, appeared too high to be recommended for the developmental toxicity study in rabbits. The 10 mg/kg bw/day stomach tube dosage would be expected to be a no-observable-adverse-effect-level (NOAEL) for both maternal and embryo-foetal toxicity and the 40 mg/kg bw/day stomach tube dosage would be expected to produce maternal toxicity and minimal developmental toxicity. As comparable toxicity occurred via both the dietary and stomach tube routes, the gavage route was used in the study based on the ability to more accurately achieve the desired dosage.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
Examination for clinical observations of effects of the test substance, abortions, premature deliveries and deaths

BODY WEIGHT: Yes
- Time schedule for examinations: recorded on DGs 0 and 6 through 29

FOOD CONSUMPTION AND COMPOUND INTAKE : Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined:
The thoracic, abdominal and pelvic viscera of each rabbit were examined for gross lesions. Uteri of rabbits that appeared nonpregnant were stained with 10% ammonium sulfide, to confirm the absence of implantation sites. Gross lesions were preserved in neutral buffered 10% formalin for possible future evaluation (with the exception of parovarian cysts, which are common, spontaneous lesions in rabbits); all other tissues were discarded.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [all per litter]

Statistics:

Bartlett’s test, Fisher's exact test, Dunnet’s test, Kruskall-Wallis test; Dunn’s test

Historical control data:

Yes

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The 40 mg/kg bw/day dosage group had significantly increased numbers of rabbits with abnormal faeces. Scant faeces and no faeces were observed for one or more does on DGs 7 to 24 and DGs 9 to 17, respectively. All other clinical observations were considered unrelated to the test substance because the incidences were not dosage-related.

Mortality:

no mortality observed

Description (incidence):

No deaths or abortions occurred during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Maternal body weight gain for the entire dosage period was reduced in the 20 mg/kg/day dosage group (-12 %, p<0.05) and in the 40 mg/kg/day dosage group (-76%, p<0.01), as compared with the control group values. Within this period, the 20 mg/kg/day dosage group had significantly reduced weight gain on DGs 12 to 15 (-56%), and the 40 mg/kg/day dosage group had significant weight losses on DGs 6 to 12 (from -238% to -233%) and significantly reduced weight gain on DGs 12 to 15 (-67%). Reflecting these effects of the test substance, weight gains for the entire gestation period were significantly reduced in the 40 mg/kg/day dosage group (-68%). Maternal body weights were generally significantly reduced in the 40 mg/kg/day dosage group on DGs 10 to 29 (from -8% to -6%).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Absolute and relative feed consumption values were significantly reduced in the 20 and 40 mg/kg/day dosage groups for the entire dosage period (-12% and -12% at 20 mg/kg/day; -44% and -41 % at 40 mg/kg/day). Within this period, relative feed consumption values were significantly reduced in the 20 mg/kg/day dosage group on DGs 6 to 18 (from -20% to -16%), and absolute and relative feed consumption values were significantly reduced in the 40 mg/kg/day dosage group on DGs 6 to 24 (absolute, from -71 % to -15 %) or 21 (relative, from -70% to -24%).

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

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The only necropsy observations were parovarian cysts occurring for one control group doe and four 10 mg/kg bw/day dosage does. The statistically significant increase in this gross lesion in the 10 mg/kg bw/day dosage group was not considered related to the test substance because 1) the incidence was not dosage-dependent and 2) this is a common observation in pregnant rabbits.

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:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

One control group litter consisted of two early resorptions, an event that is not unusual for a litter of only two conceptuses. Because such events may abnormally skew the data distributions, all values for this doe and litter were excluded from statistical analyses.

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

One 5 mg/kg bw/day dosage group doe had a litter consisting of eight live foetuses, two dead foetuses and one late resorption, a non-dosage dependent observation. No other dead foetuses occurred in this study.

Changes in pregnancy duration:

effects observed, non-treatment-related

Description (incidence and severity):

Two premature deliveries occurred that were considered unrelated to effects of the test substance as they occurred in the 10 and 20 mg/kg bw/day dose groups and thus were not dosage-related.

Changes in number of pregnant:

no effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

maternal

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

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

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One 5 mg/kg bw/day dosage group fetus had an umbilical hernia with protrusion of the intestines. One 20 mg/kg bw/day dosage group fetus had a skin closure defect on the head and short 3rd and 4th digits on the right hindpaw with absence of the 3rd
and 4th phalanges (first observed at skeletal examination). No gross external variations occurred in the fetuses in this study.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

The 40 mg/kg bw/day dosage group had a significant increase in the average for thoracic ribs (supernumerary ribs), with associated significant increases and reductions in the average for thoracic and lumbar vertebrae, respectively. This variation was considered an effect of the test substance because the values were statistically significant and exceeded the historical ranges of the testing facility. This variation in rib development was considered by the study authors to be a common finding at maternally toxic dosages and to be reversible.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One 40 mg/kg/day dosage group fetus had a diaphragmatic hernia as the only fetal alteration. The stomach and the left lateral lobe and a portion of the median lobe of the liver protruded through the hernia and into the left dorsal quadrant thoracic cavity.
One 40 mg/kg/day dosage group fetus had a pink 0.3 cm x 0.1 cm mass present on the cortex of the left kidney.
One control group fetus had fused apical and cardiac lung lobes and absence of the left diaphragmatic lung lobe. One control group fetus, one 5 mg/kg bw/kg/day dosage group fetus, two 10 mg/kg bw/day dosage group littermates, two 20 mg/kg bw/day dosage group littermates and two fetuses from different 40 mg/kg bw/day dosage litters had absence of the intermediate lung lobe, a common variation in lung development in this rabbit strain. No additional alterations occurred in these fetuses.
One 5 mg/kg bw/day dosage group fetus had a circumcorneal hemorrhage in one eye, a variation generally attributable to trauma during processing.
One 5 mg/kg bw/day dosage group fetus had no gallbladder as the only fetal alteration.

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: supernumerary rib

Description (incidence and severity):

This variation in rib development was considered by the study authors to be a common finding at maternally toxic dosages and to be reversible.

Key result

Developmental effects observed:

no

Conclusions:

The NOAEL for maternal toxicity was 10 mg/kg bw/day based on reductions in maternal body weight gain and absolute and relative feed consumption. The developmental NOAEL was set at 20 mg/kg bw/day based on supernumerary thoracic ribs at 40 mg/kg bw/day.

Executive summary:

In a study according OECD TG 414 pregnant New Zealand White rabbits were used to investigate the teratogenic potential of the test item.

Dosages of 0, 5, 10, 20 or 40 mg/kg bw/day were investigated, administered by stomach tube to pregnant rabbits on GDs 6 through 28, with Caesarean-delivery of the foetuses on GD 29. All rabbits were observed for viability at least twice each day of the study. Additional examinations for clinical observations of effects of the test substance, abortions, premature deliveries and deaths were made before each daily intubation and approximately 60 minutes after intubation during dosage period. These observations were also made on the day the rabbits were sacrificed (DG29). Body weights were recorded on DGs 0 and 6 through 29. Feed consumption values were recorded daily after arrival at the Testing Facility. All rabbits were sacrificed on DG 29. The thoracic, abdominal and pelvic viscera of each rabbit were examined for gross lesions. The number of corpora lutea in each ovary was recorded. The uterus was excised and examined for pregnancy, number and distribution of implantations, early and late resorptions and live and dead foetuses. Each Caesarean-delivered foetus was weighed, examined for gross external alterations and individually identified with a tag noting study number, litter number and uterine distribution. After sacrifice, all live foetuses were examined internally to identify sex and visceral alterations; cavitated organs were evaluated by dissection. The brain was cross-sectioned and examined in situ. All foetuses were evaluated for skeleton alterations. Rabbits that prematurely delivered a litter were evaluated following the procedures described above. Delivered pups were weighed, examined for sex, and external and visceral alterations. Pups delivered on the day of scheduled Caesarean-sectioning were also examined for skeletal alterations.

No mortality was observed. The 40 mg/kg bw dosage group had significantly increased numbers of rabbits with abnormal faeces. Scant faeces and no faeces were observed for one or more does on DGs 7 to 24 and DGs 9 to 17, respectively. All other clinical observations were considered unrelated to the test substance because the incidences were not dosage-related.

Maternal body weight gain for the entire dosage period was reduced in the 20 mg/kg/day dosage group (-12 %, p<0.05) and in the 40 mg/kg/day dosage group (-76%, p<0.01), as compared with the control group values. Within this period, the 20 mg/kg/day dosage group had significantly reduced weight gain on DGs 12 to 15 (-56%), and the 40 mg/kg/day dosage group had significant weight losses on DGs 6 to 12 (from -238% to -233%) and significantly reduced weight gain on DGs 12 to 15 (-67%). Reflecting these effects of the test substance, weight gains for the entire gestation period were significantly reduced in the 40 mg/kg/day dosage group (-68%). Maternal body weights were generally significantly reduced in the 40 mg/kg/day dosage group on DGs 10 to 29 (from -8% to -6%).

Absolute and relative feed consumption values were significantly reduced in the 20 and 40 mg/kg/day dosage groups for the entire dosage period (-12% and -12% at 20 mg/kg/day; -44% and -41 % at 40 mg/kg/day). Within this period, relative feed consumption values were significantly reduced in the 20 mg/kg/day dosage group on DGs 6 to 18 (from -20% to -16%), and absolute and relative feed consumption values were significantly reduced in the 40 mg/kg/day dosage group on DGs 6 to 24 (absolute, from -71 % to -15 %) or 21 (relative, from -70% to -24%). At 40 mg/kg/day scant faeces or no faeces were also observed.

Concentration and homogeneity analyses indicated no test substance contamination of the vehicle, all samples for the 0.5, 1.0, 2.0 and 4.0 mg/ml concentrations were within ±1 0% of target (5, 10, 20 and 40 mg/kg/day dosages, respectively) and the formulations were homogeneous. Stability analyses indicated the formulations were stable for a period of at least 7 days when stored refrigerated.

No abortions occurred during the study. Two premature deliveries occurred that were considered unrelated to effects of the test substance as they occurred in the 10 and 20 mg/kg bw/day dose groups and thus were not dosage-related. One control group litter consisted of two early resorptions, an event that is not unusual for a litter of only two conceptuses. Because such events may abnormally skew the data distributions, all values for this doe and litter were excluded from statistical analyses. One 5 mg/kg/day dosage group doe had a litter consisting of eight live fetuses, two dead fetuses and one late resorption, as previously described a non-dosage dependent observation. No other dead fetuses occurred in this study.

The litter averages for corpora lutea, implantations, litter sizes, live and dead fetuses, early and late resorptions, percent resorbed conceptuses, percent male fetuses and fetal body weights, as well as the numbers of does with any resorptions or with viable fetuses did not differ significantly in the five dosage groups and were within the historical ranges of the testing facility. All placentae appeared normal.

No test substance-related fetal malformations were observed, however the 40 mg/kg/day dosage group had a significant increase in the average for thoracic ribs (supernumerary ribs), with associated significant increases and reductions in the average for thoracic and lumbar vertebrae, respectively. This variation was considered an effect of the test substance because the values were statistically significant and exceeded the historical ranges of the testing facility. This variation in rib development was considered by the study authors to be a common finding at maternally toxic dosages and to be reversible. The NOAEL for maternal toxicity was 10 mg/kg bw/day based on reductions in maternal body weight gain and absolute and relative feed consumption. The developmental NOAEL was set at 20 mg/kg bw/day based on supernumerary thoracic ribs at 40 mg/kg bw/day.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 1980 - April 1981

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: COBS® CD

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Canadian Breeding Farm and Laboratories, St. Constant, Quebec
- Age at study initiation: approximately 17 weeks
- Weight at study initiation: ca. 286 g
- Fasting period before study: no
- Housing: individually housed, except during the mating period, in suspended wire-mesh cages
- Diet: ad libitum, Purina® Certified Rodent Chow® #5002
- Water: ad libitum, tap water
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): temperature-controlled environment
- Humidity (%): humidity-controlled environment
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 5% aqueous Arabic gum

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test article was prepared at concentrations to permit administration at dosage levels of 100, 300 and 1000 mg/kg bw/day at a constant volume of 10 mL/kg. A magnetic stir bar and plate were used to ensure adequate suspension during administration.

VEHICLE
- Justification for use and choice of vehicle: Substance no-soluble in water.
- Concentration in vehicle: 5%
- Amount of vehicle: 10 mL/kg

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1:1
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: plug or a vaginal smear for sperm

Duration of treatment / exposure:

days 6 through 19 of gestation

Frequency of treatment:

daily

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
COBS CD rats were used in the range-finding study (animals 12 weeks old, breeder: Charles River, Portage Michigan, USA). In the range-finding study, the dose levels were 0; 250; 500; 1000; 3000 and 5000 mg/kg bw/day. The test-item was administered by gavage to 5 animals per dose group. The administration was carried out at a constant volume of 10 mL/kg bw from days 6 through 19 of gestation. No mortality or dose-related signs of clinical toxicity were noted. Dose-related reductions in mean maternal body weight gain which ranged from slight to severe occurred in animals treated with doses of 1000 mg/kg bw/day and higher. There were no dose-related trends in the mean numbers of corpora lutea, total implantations, viable foetuses or mean post implantation losses. A slight increase in the number of early resorptions was noted in the 500 and 3000 mg/kg bw/day groups (1.2 and 1.4 per litter, respectively; control group 0.3 per litter). However, as a similar increase was not observed in the 5000 mg/kg bw/day group (0.0 per litter), this effect was not considered to be treatment related.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
Animals observed daily for mortality and clinical signs of toxicity on days 6 through 20 of gestation.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual maternal body weights were recorded on gestation days 0, 6, 9, 12, 16 and 20.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uterus was excised and weighed prior to removal of the fetuses, abdominal and thoracic organs and cavities of the dams were examined for grossly evident morphological changes and the carcasses discarded. Uteri from females that appeared nongravid were opened and placed in an approximate 10% ammonium sulfide solution for confirmation of pregnancy status.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [ half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: No

Statistics:

Chi-square test, Fisher's exact test, Mann-Whitney U test, Bartlett's test, Dunnett's test

Historical control data:

yes

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no biologically meaningful differences in the appearance or behavior of rats in any of the treatment groups when compared to the control group. Hair loss, primarily of the limbs and ventral abdominal and dorsal posterior regions occurred with similar frequency in all groups. Soft stool was observed in three rats in the control group and one rat in the 1000 mg/kg/day treatment group at various times during the treatment period.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At the high dose, there was no body weight gain over the first three days of treatment and therefore a slightly reduced body weight gain was noted at the end of the study (-8% of control group, day 0-20). This also concerned the adjusted body weight gain (-22% of control group, day 0-20).

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Postmortem observations at the time of Cesarean section included one rat each in the control, 100 and 1000 mg/kg bw/day treatment groups and two in the 300 mg/kg bw/day treatment group with bilateral hydrometra. Multiple pinpoint (1 mm) red foci were noted on the thymus of one rat in the control group. Also in the control group, one rat had two bladder stones (6 mm and 8 mm in diameter) along with distended ureters and one kidney with hydronephrosis and pitting. One rat in the 100 mg/kg bw/day treatment group had bilateral hydronephrosis. One rat in the 300 mg/kg bw/day treatment group had a cyst (1 mm diameter) on one kidney.

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:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

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:

maternal

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

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

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

There were no biologically meaningful or statistically significant differences in the total number of litters with malformations in any of the treatment groups when compared to the control group.

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

NOAEL for developmental toxicity was 1000 mg/kg bw/day. NOAEL for maternal toxicity was 300 mg/kg bw/day based on reductions in body weight gain at 1000 mg/kg bw/day. The substance is not teratogenic to rats under the conditions of the study.

Executive summary:

A study according OECD TG 414 was conducted to assess the developmental toxicity of the substance in rats. COBS CD rats were used (animals 17 weeks old). The substance was administered by gavage to 25 animals per dose group. The administration was carried out at a constant volume of 10 mL/kg bw from days 6 through 19 of gestation. The dose levels administered in the main study were 0; 100; 300 and 1000 mg/kg bw/day. The control group in each study received only the vehicle, 5% aqueous Arabic gum. Uterine examinations were performed on all animals on day 20 of gestation.

The administration of the test substance did not result in mortalities or in any clinical signs of toxicity. At the high dose, there was no body weight gain over the first three days of treatment and therefore a slightly reduced body weight gain was noted at the end of the study (-8% of control group, day 0-20). This also concerned the adjusted body weight gain (-22% of control group, day 0-20).

The assessment of the mean numbers of corpora lutea, total implantations, post implantation loss, viable fetuses, mean fetal body weight or fetal sex distribution revealed no test substance related adverse effects.

The external, visceral and skeletal examination of all fetuses revealed no teratogenic effects.

NOAEL for developmental toxicity was 1000 mg/kg bw/day. NOAEL for maternal toxicity was 300 mg/kg bw/day based on reductions in body weight gain at 1000 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

20 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

OECD TG 414

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Toxicity to reproduction: other studies

Additional information

Developmental toxicity rats

Key

A study according OECD TG 414 was conducted to assess the developmental toxicity of the substance in rats. COBS CD rats were used (animals 17 weeks old). The substance was administered by gavage to 25 animals per dose group. The administration was carried out at a constant volume of 10 mL/kg bw from days 6 through 19 of gestation. The dose levels administered in the main study were 0; 100; 300 and 1000 mg/kg bw/day. The control group in each study received only the vehicle, 5% aqueous Arabic gum. Uterine examinations were performed on all animals on day 20 of gestation.

The administration of the test substance did not result in mortalities or in any clinical signs of toxicity. At the high dose, there was no body weight gain over the first three days of treatment and therefore a slightly reduced body weight gain was noted at the end of the study (-8% of control group, day 0-20). This also concerned the adjusted body weight gain (-22% of control group, day 0-20).

The assessment of the mean numbers of corpora lutea, total implantations, post implantation loss, viable fetuses, mean fetal body weight or fetal sex distribution revealed no test substance related adverse effects.

The external, visceral and skeletal examination of all fetuses revealed no teratogenic effects.

NOAEL for developmental toxicity was 1000 mg/kg bw/day. NOAEL for maternal toxicity was 300 mg/kg bw/day based on reductions in body weight gain at 1000 mg/kg bw/day.

Developmental toxicity rabbits

Key

In a study according OECD TG 414 pregnant New Zealand White rabbits were used to investigate the teratogenic potential of the test item.

Dosages of 0, 5, 10, 20 or 40 mg/kg bw/day were investigated, administered by stomach tube to pregnant rabbits on GDs 6 through 28, with Caesarean-delivery of the foetuses on GD 29. All rabbits were observed for viability at least twice each day of the study. Additional examinations for clinical observations of effects of the test substance, abortions, premature deliveries and deaths were made before each daily intubation and approximately 60 minutes after intubation during dosage period. These observations were also made on the day the rabbits were sacrificed (DG29). Body weights were recorded on DGs 0 and 6 through 29. Feed consumption values were recorded daily after arrival at the Testing Facility. All rabbits were sacrificed on DG 29. The thoracic, abdominal and pelvic viscera of each rabbit were examined for gross lesions. The number of corpora lutea in each ovary was recorded. The uterus was excised and examined for pregnancy, number and distribution of implantations, early and late resorptions and live and dead foetuses. Each Caesarean-delivered foetus was weighed, examined for gross external alterations and individually identified with a tag noting study number, litter number and uterine distribution. After sacrifice, all live foetuses were examined internally to identify sex and visceral alterations; cavitated organs were evaluated by dissection. The brain was cross-sectioned and examined in situ. All foetuses were evaluated for skeleton alterations. Rabbits that prematurely delivered a litter were evaluated following the procedures described above. Delivered pups were weighed, examined for sex, and external and visceral alterations. Pups delivered on the day of scheduled Caesarean-sectioning were also examined for skeletal alterations.

No mortality was observed. The 40 mg/kg bw dosage group had significantly increased numbers of rabbits with abnormal faeces. Scant faeces and no faeces were observed for one or more does on DGs 7 to 24 and DGs 9 to 17, respectively. All other clinical observations were considered unrelated to the test substance because the incidences were not dosage-related.

Maternal body weight gain for the entire dosage period was reduced in the 20 mg/kg/day dosage group (-12 %, p<0.05) and in the 40 mg/kg/day dosage group (-76%, p<0.01), as compared with the control group values. Within this period, the 20 mg/kg/day dosage group had significantly reduced weight gain on DGs 12 to 15 (-56%), and the 40 mg/kg/day dosage group had significant weight losses on DGs 6 to 12 (from -238% to -233%) and significantly reduced weight gain on DGs 12 to 15 (-67%). Reflecting these effects of the test substance, weight gains for the entire gestation period were significantly reduced in the 40 mg/kg/day dosage group (-68%). Maternal body weights were generally significantly reduced in the 40 mg/kg/day dosage group on DGs 10 to 29 (from -8% to -6%).

Absolute and relative feed consumption values were significantly reduced in the 20 and 40 mg/kg/day dosage groups for the entire dosage period (-12% and -12% at 20 mg/kg/day; -44% and -41 % at 40 mg/kg/day). Within this period, relative feed consumption values were significantly reduced in the 20 mg/kg/day dosage group on DGs 6 to 18 (from -20% to -16%), and absolute and relative feed consumption values were significantly reduced in the 40 mg/kg/day dosage group on DGs 6 to 24 (absolute, from -71 % to -15 %) or 21 (relative, from -70% to -24%). At 40 mg/kg/day scant faeces or no faeces were also observed.

Concentration and homogeneity analyses indicated no test substance contamination of the vehicle, all samples for the 0.5, 1.0, 2.0 and 4.0 mg/ml concentrations were within ±1 0% of target (5, 10, 20 and 40 mg/kg/day dosages, respectively) and the formulations were homogeneous. Stability analyses indicated the formulations were stable for a period of at least 7 days when stored refrigerated.

No abortions occurred during the study. Two premature deliveries occurred that were considered unrelated to effects of the test substance as they occurred in the 10 and 20 mg/kg bw/day dose groups and thus were not dosage-related. One control group litter consisted of two early resorptions, an event that is not unusual for a litter of only two conceptuses. Because such events may abnormally skew the data distributions, all values for this doe and litter were excluded from statistical analyses. One 5 mg/kg/day dosage group doe had a litter consisting of eight live fetuses, two dead fetuses and one late resorption, as previously described a non-dosage dependent observation. No other dead fetuses occurred in this study.

The litter averages for corpora lutea, implantations, litter sizes, live and dead fetuses, early and late resorptions, percent resorbed conceptuses, percent male fetuses and fetal body weights, as well as the numbers of does with any resorptions or with viable fetuses did not differ significantly in the five dosage groups and were within the historical ranges of the testing facility. All placentae appeared normal.

No test substance-related fetal malformations were observed, however the 40 mg/kg bw/day dosage group had a significant increase in the average for thoracic ribs (supernumerary ribs), with associated significant increases and reductions in the average for thoracic and lumbar vertebrae, respectively. This variation was considered an effect of the test substance because the values were statistically significant and exceeded the historical ranges of the testing facility. This variation in rib development was considered by the study authors to be a common finding at maternally toxic dosages and to be reversible. The NOAEL for maternal toxicity was 10 mg/kg bw/day based on reductions in maternal body weight gain and absolute and relative feed consumption. The developmental NOAEL was set at 20 mg/kg bw/day based on supernumerary thoracic ribs at 40 mg/kg bw/day.

Conclusion

In the RAC opinion on harmonized classification and labelling (15th March 2019) the skeletal effects which have been observed in the OECD TG 414 in rabbits were evaluated as secondary to maternal toxicity and are considered as variations. In conclusion, RAC is of the opinion that the available data are conclusive and that classification of the test item for developmental toxicity is not warranted.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available information is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available experimental information, the test substance is not classified for toxicity to reproduction or developmental toxicity according to Regulation (EC) No 1272/2008 (CLP), as amended for the seventeenth time in Regulation (EU) 2021/849.