Fenamiphos

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Endpoints for toxicity to reproduction are covered by available animal studies.

 

The test substance did not show adverse effects on reproduction.

 

The NOAEL for reproductive toxicity was 2.8 mg/kg bw/day in a 2-generation study in rats, based on the absence of reproductive effects at the highest dose tested.

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1989-02-08 to 1991-04-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

May 1983

Deviations:

no

GLP compliance:

yes

Limit test:

no

Justification for study design:

This study was conducted to provide general information concerning the effects of the test substance on the reproductive organs, estrous cycles, mating behavior, conception, parturition, lactation, and offspring growth and development.

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

standard species

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Sasco Inc., Omaha, Nebraska
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) males: 8 weeks old, females 6 weeks old; (F1) x wks
- Weight at study initiation: (P) Males: 227 - 233 g; Females: 158 - 161 g; (F1) Males: 176 - 200 g; Females: 140.3 - 156.0 g
- Fasting period before study: none
- Housing:
During pre-mating periods, rats were housed individually in stainless steel cages suspended over deotized animal cage board bedding. Pregnant females were housed individually in polycarbonate plastic cages with corn cob bedding (Bed-o-Cobs).
- Diet (ad libitum): Purina Rodent Laboratory Chow 5001-4 Etts form
- Water (ad libitum): potable municipal
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26
- Humidity (%): 40 - 70
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1989-01-31 To: 1989-11-06

Route of administration:

oral: feed

Vehicle:

corn oil

Details on exposure:

DIET PREPARATION
The test substance was dissolved in corn oil and mixed in the diet. The corn oil was added at 1% of the diet for all dose groups. Acetone, a highly volatile solvent, was used in diet preparation for rinsing equipment.

- Rate of preparation of diet: weekly

- Storage temperature of food: -23 °C

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: up to 21 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear examined microscopically for sperm referred to as day 0 of pregnancy
- Unmated females were then co-housed for up to seven days during a fourth week of breeding with a proven male from the same dose group.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: in a polycarbonate cage
for gestation and lactation.

Duration of treatment / exposure:

From initiation to sacrifice.
Before mating P0 and F1: 10 weeks

Frequency of treatment:

not applicable, feed ad libitum

Dose / conc.:

0 mg/kg diet

Dose / conc.:

2.5 mg/kg diet

Dose / conc.:

10 mg/kg diet

Dose / conc.:

40 mg/kg diet

No. of animals per sex per dose:

30 animals per dose

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The doses for this study were selected from previous studies, and discussed with and agreed upon by the U.S. EPA.

- Rationale for animal assignment:
Parents (FO) for the Fl generation were assigned to dose groups based on weight using software from Instem Computer Systems Pic., Stone, Staffordshire, U.K. Pups to be culled on day 4 postpartum and F1a weanlings to become parents (Fl) for the F2 generation were selected at random using SAS software (SAS Institute Inc., Gary, North Carolina). For F1 parents the genealogy of the Fla pups was checked to prevent the mating of litter mates.

- Fasting period before blood sampling for clinical biochemistry: not specified

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations:
During the ten-week period prior to the P0 and F1 matings, male and female body weights and feed consumption were measured once a week. Body weights and food consumption were taken for dams only during gestation and lactation as follows: gestation - body weight on days 0, 6, 13, and 20; food consumption once a week; lactation - body weight on days 1, 4, 7, 14, and 21; food consumption twice during week 1 and once a week during weeks 2 and 3.

FOOD CONSUMPTION AND COMPOUND INTAKE: weekly, see above

OTHER: CHOLINESTERASE
Cholinesterase activity was determined.
Plasma Cholinesterase (PCHE), red blood cell Cholinesterase (RCHE) and brain Cholinesterase (BCHE) were determined for ten F0 and F1 adults/sex/dose group. The PCHE and RCHE were measured during week 8 of the F1a and F2a premating periods and just prior to sacrifice. The BCHE was measured at termination. The PCHE, RCHE and BCHE were determined for one male and one female pup from each of ten litters/dose group at the time of culling (i.e., day 4 postpartum) and at weaning (i.e., day 21 postpartum). Pooling of blood from four-day old pups of the same sex within a litter was occasionally done to obtain sufficient blood for the analyses. When blood was pooled, the brains from these animals were also pooled for analysis.

Oestrous cyclicity (parental animals):

Vaginal smears were taken for two weeks from ten FO and Fl females/dose level prior to mating. The vaginal smears were observed microscopically and classified into one of the following four categories:
L = Leukocyte: Five or more leukocytes in a field indicating diestrus.
P = Proestrus: Large rounded/nucleated cells indicating proestrus.
P/C = Proestrus/Cornified: A transition phase from proestrus to estrus.
C = Cornified cells: Large serrated cells indicating estrus.

This procedure was done to characterize the estrous cycle and to determine if females in each dose level were cycling properly.

Sperm parameters (parental animals):

Parameters examined in all male parental generations:
The testes, pituitary, epididymides, seminal vesicles/ coagulating gland, prostate gland, and gross lesions were collected and fixed in Bouin's fixative.

Litter observations:

The number of live and stillborn pups were recorded for each litter. Litter counts were performed from days 0 to 21. Individual pup weights were recorded at birth (day 0) and on days 4, 7, 14, and 21. The size of each litter was adjusted (culled) on day 4 to yield eight pups, including if possible, four males and four females per litter. No adjustment was made in litters of eight or fewer pups. Adjustments were made by the random selection of pups from each litter.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were sacrificed and necropsied after the last F1 and F2 litters, respectively, were delivered or dams had gone past day 24 of gestation.
- Maternal animals: All surviving animals were sacrificed and necropsied after each dam's pups were weaned or when day 24 of gestation was reached.

GROSS NECROPSY: yes

HISTOPATHOLOGY / ORGAN WEIGHTS
Females: Ovary and terminal body weights were measured. The pituitary, vagina, cervix, uterus, ovaries, and gross lesions were collected and fixed in Bouin's fixative. The uterus from all dams was examined for implantation sites, and the number of sites were counted. The above tissues and gross lesions were embedded in paraffin, sectioned, and stained with hematoxylin-eosin for histological examination.

Postmortem examinations (offspring):

- Cholinesterase determination
- Gross necropsy
- Other: For animals found dead on day 0 postpartum, the ability of the lungs to float in water was evaluated to determine if the pups were stillborn (i.e., the lungs of pups born alive float).

Statistics:

Body weight, food consumption, organ weights, and cholinesterase data* were analyzed by analysis of variance (ANOVA). If significant differences were shown by ANOVA, Dunnett's test was used to identify significant differences from the control group. Litter size, length of gestation, viability indices, birth index, live birth index, percent of male and female pups, time required for insemination, and number of implantation sites were analyzed by the Kruskal-Wallis test. If significant differences were shown by Kruskal-Wallis, the Mann-Whitney test was used to identify statistical significance between groups. The mating index, fertility index, gestation index, and number of litters with stillborn and unknown alive/dead pups were analyzed using the Chi-square test. If significant differences were shown by Chi-square, FISHER'S exact test was used to identify statistical significance between groups. However, if 50% of the cells in the Chi-square test had expected counts less than 5, the above parameters were evaluated using FISHER'S exact test with a Bonferroni adjustment of the p value (i.e., adjusted p = p/number of comparisons). Histopathologic lesion frequencies were examined for unusual patterns. Those with either obvious or uncertain (in the opinion of the pathologist) significance were examined statistically using FISHER'S exact test. Statistical significance was determined at p<0.05 for all tests unless otherwise noted.

Reproductive indices:

The following indices were calculated for each dose group: mating index, fertility index, gestation index on days 4, 7, 14 and 21.

Offspring viability indices:

The following indices were calculated for each dose group: birth index, live birth index, and viability index on days 4, 7, 14 and 21.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 40 mg/kg food, statistically significant reductions were seen in body-weight gain during lactation (by 72 %) and food consumption (by up to 11 %).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

An incidental finding was a slight (5%), but statistically significant, decrease in female food consumption on day 7 for the low- and mid-dose groups.

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Plasma ChE activity was significantly inhibited by > 20% in all treated adult females of both generations, in F0 males at 40 mg/kg food both before mating and at sacrifice. Erythrocyte ChE activity was significantly inhibited in females at doses ≥ 10 mg/kg food but only at 40 mg/kg food in F0 males. At 40 mg/kg food, brain ChE activity was significantly inhibited in F0 females.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

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):

40 mg/kg food: increase in oedema of the salivary glands in both males and females

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

0.17 - 0.2 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

10 mg/kg diet

System:

nervous system

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In F1 dams at 40 mg/kg food, statistically significant reductions were seen in body-weight gain during lactation (by 65%) and food consumption (by up to 19%). The body weights of F1 adults at 40 mg/kg food were significantly reduced throughout the premating period (by about 10% in males and 7% in females). The overall weight gain of F1 males before mating was also reduced during the first four weeks, by 12% at 10 mg/kg food and 15% at 40 mg/kg food.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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):

Plasma ChE activity was significantly inhibited by > 20% in all treated adult females of both generations, in F1 males at 10 mg/kg food at the time of sacrifice, and in F1 males at 40 mg/kg food both before mating and at sacrifice. Erythrocyte ChE activity was significantly inhibited in females at doses ≥ 10 mg/kg food but only at 40 mg/kg food in F1 males. At 40 mg/kg food, brain ChE activity was significantly inhibited in F1 females.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

0.17 - 0.2 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

40 mg/kg food: decrease in pup weight gain in lactation periodd

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There was a treatment-related decrease in food consumption in the high-dose group on days 14 and 21 of the F1a lactation period (11 % and 8 %, respectively; statistically significant on day 14).

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

see "Food consumption and compound intake (if feeding study)"

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 pups, plasma ChE activity was significantly inhibited on day 21 of lactation in both males and females of both generations at doses ≥10 mg/kg food. Erythrocyte ChE activity was inhibited by > 20 % in males and females of both generations at 40 mg/kg food on day 21. Brain ChE activity was not affected in pups.

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:

no effects observed

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

clinical biochemistry

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

40 mg/kg food: decrease in pup weight gain in lactation periodd

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

40 mg/kg food: There was a treatment-related decrease in food consumption throughout the F2a lactation period (11% to 19%; statistically significant at all time periods).

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

see "Food consumption and compound intake (if feeding study)"

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There was no statistical significant depression in PCHE, RCHE or BCHE in 4-day old pups, except for a slight (11.8%) decrease in PCHE in 4-day old F2a female pups in the 40 ppm dose group which was not considered biologically significant.

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:

no effects observed

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

nervous system

Organ:

other: ChE

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Reproductive effects observed:

no

Conclusions:

The NOAEL for parental toxicity was 2.5 mg/kg food, equal to 0.17 mg/kg bw/day, on the basis of decreased body-weight gain in males and inhibition of erythrocyte ChE activity in both males and females. The NOAEL for developmental toxicity was 10 mg/kg food, equal to 0.64 mg/kg bw/day, on the basis of decreased pup body weights during lactation and in addition inhibition of erythrocyte ChE activity. The NOAEL for reproductive toxicity was 40 mg/kg food, equal to 2.8 mg/kg bw/day, based on the absence of reproductive effects at the highest dose tested.

Executive summary:

The test substance was administered via the diet to CD Sprague-Dawley rats for two generations to test for potential reproductive effects. The test compound was administered at nominal dose levels of 0, 2.5, 10, and 40 ppm (actual doses were 2,40, 9.12, and 38.9 ppm). The test item given in the diet at concentrations of 0, 2.5, 10, or 40 ppm (mg/kg food) for 70 days before mating was equal to doses of 0.17, 0.64, or 2.8 mg/kg bw/day for males and for females 0.20, 0.73, or 3.2 mg/kg bw/day in the premating period, 0.17, 0.64, and 2.82 mg/kg bw/day in the gestation period and 0.39, 1.48, 5.85 mg/kg bw/day in the lactation period.

The F0 and F1 parents were comprised of 30 rats/sex/ group. The F0 and F1 parents received the test item in the diet throughout the entire study, beginning at six and eight weeks of age for female and male F0 parents, respectively, and at weaning for the F1 parents. Prior to breeding, the animals received treated feed for a ten week period. F0 parents were mated to produce F1a litters and F1 parents (F1a pups randomly selected to be F1 parents) were mated to produce F2a litters.

General findings: No treatment-related clinical findings and deaths were observed in the parents or pups.

Treatment-related lower body weights were observed in the F1 high-dose group animals during the premating (males and females) and gestation (females) periods, however, no difference in gestation weight.

gain was observed. The lower body weights observed during the premating and gestation periods were due to a decrease in F1a pup body weight gain during the lactation period (F1a pups = F1 adults).

During the F1a and F2a lactation phases, lower body weights were observed in F0 and F1 high-dose group females. The lower body weights correlated with a decrease in food consumption during the lactation periods.

Reproduction: Oestrous cycles, mating, fertility, and gestation indices, sex ratio, and pup viability indices were unaffected at 40 ppm. There were no treatment-related effects on pup development in the low- and middle-dose groups. A decrease in pup weight gain in the high dose group was observed in the F1a and F2a generation.

Cholinesterase (ChE) measurements: Plasma ChE activity was lower at > 2.5 ppm in all adult females of both generations, in F1 males at > 10 ppm and in F0 males at 40 ppm. Ery ChE activity was lower in females at > 10 ppm and in males at 40 ppm. Brain ChE activity was lower at 40 ppm in F0 males and females and in F1 females. In 4 day old pups plasma ChE was lower at 40 ppm; ery and brain ChE were not affected. In 21 day old pups plasma ChE was lower at > 10 ppm and ery ChE at 40 ppm; brain ChE was not affected.

Gross pathology, organ weights, histopathology: No treatment-related gross or microscopic lesions were observed.

The NOAEL for parental toxicity was 2.5 mg/kg food, equal to 0.17 mg/kg bw/day, on the basis of decreased body-weight gain in males and inhibition of erythrocyte ChE activity in both males and females. The NOAEL for developmental toxicity was 10 mg/kg food, equal to 0.64 mg/kg bw/day, on the basis of decreased pup body weights during lactation and in addition inhibition of erythrocyte ChE activity. The NOAEL for reproductive toxicity was 40 mg/kg food, equal to 2.8 mg/kg bw/day, based on the absence of reproductive effects at the highest dose tested.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

Study duration:

subchronic

Species:

rat

Quality of whole database:

Klimisch 1

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The information provided below was taken from the original plant protection dossier on the active substance submitted in 2017 for inclusion of the test substance to Annex I of Directive 91/414/EEC and has been previously evaluated in the Draft Assessment Report (DAR) according to the Commission Regulation (EU) No 1107/2009 (2003; Renewal Assessment Report (RAR) 3rd Revision: 2018)) and subject to peer review by EFSA and Member States (2006).

 

Eigenberg (1991): In a study according to OECD No. 416, the test substance was administered via the diet to CD Sprague-Dawley rats for two generations to test for potential reproductive effects. The test compound was administered at nominal dose levels of 0, 2.5, 10, and 40 ppm (actual doses were 2,40, 9.12, and 38.9 ppm). The test item given in the diet at concentrations of 0, 2.5, 10, or 40 ppm (mg/kg food) for 70 days before mating was equal to doses of 0.17, 0.64, or 2.8 mg/kg bw/day for males and for females 0.20, 0.73, or 3.2 mg/kg bw/day in the premating period, 0.17, 0.64, and 2.82 mg/kg bw/day in the gestation period and 0.39, 1.48, 5.85 mg/kg bw/day in the lactation period. The F0 and F1 parents were comprised of 30 rats/sex/ group. The F0 and F1 parents received the test item in the diet throughout the entire study, beginning at six and eight weeks of age for female and male F0 parents, respectively, and at weaning for the F1 parents. Prior to breeding, the animals received treated feed for a ten week period. F0 parents were mated to produce F1a litters and F1 parents (F1a pups randomly selected to be F1 parents) were mated to produce F2a litters. No treatment-related clinical findings and deaths were observed in the parents or pups. Treatment-related lower body weights were observed in the F1 high-dose group animals during the premating (males and females) and gestation (females) periods, however, no difference in gestation weight gain was observed. The lower body weights observed during the premating and gestation periods were due to a decrease in F1a pup body weight gain during the lactation period (F1a pups = F1 adults). During the F1a and F2a lactation phases, lower body weights were observed in F0 and F1 high-dose group females. The lower body weights correlated with a decrease in food consumption during the lactation periods. Oestrous cycles, mating, fertility, and gestation indices, sex ratio, and pup viability indices were unaffected at 40 ppm. There were no treatment-related effects on pup development in the low- and middle-dose groups. A decrease in pup weight gain in the high dose group was observed in the F1a and F2a generation. Plasma ChE activity was lower at > 2.5 ppm in all adult females of both generations, in F1 males at > 10 ppm and in F0 males at 40 ppm. Ery ChE activity was lower in females at > 10 ppm and in males at 40 ppm. Brain ChE activity was lower at 40 ppm in F0 males and females and in F1 females. In 4 day old pups plasma ChE was lower at 40 ppm; ery and brain ChE were not affected. In 21 day old pups plasma ChE was lower at > 10 ppm and ery ChE at 40 ppm; brain ChE was not affected. No treatment-related gross or microscopic lesions were observed. The NOAEL for parental toxicity was 2.5 mg/kg food, equal to 0.17 mg/kg bw/day, on the basis of decreased body-weight gain in males and inhibition of erythrocyte ChE activity in both males and females. The NOAEL for developmental toxicity was 10 mg/kg food, equal to 0.64 mg/kg bw/day, on the basis of decreased pup body weights during lactation and in addition inhibition of erythrocyte ChE activity. The NOAEL for reproductive toxicity was 40 mg/kg food, equal to 2.8 mg/kg bw/day, based on the absence of reproductive effects at the highest dose tested.

 

A summary of the results of the toxicity to reproduction study is presented in Table 1.

 

Table 1: Summary of toxicity to reproduction study results

Type of study / Reference	Species		NOAEL	LOAEL	Critical effects
			(mg/kg bw/day)	(mg/kg bw/day)
2 generation study / Eigenberg, 1991	rat	parental	0.17	0.64	Decreased body weight gain, decreased erythrocyte ChE activity
		developmental	0.64	2.8	Decreased pup body weight during lactation and decreased erythrocyte ChE activity
		reproductive	2.8	-	No effects at highest dose tested

Effects on developmental toxicity

Description of key information

Endpoints for developmental toxicity are covered by available animal studies.

 

The test substance did not show adverse effects on the development of rats or rabbits.

 

The NOAEL for developmental toxicity was 0.85 mg/kg bw/day in rats and 0.5 mg/kg bw/day in rabbits.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April - June 1986

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:

May 12, 1981

Deviations:

no

Principles of method if other than guideline:

The following deviations were noted from the revised OECD TG 414 (2001):
- The test substance was not administered until the day of scheduled kill, but only during the period of organogenesis.
- There are no summary data on gravid uterus weight.
- Only 16 female animals were used in the study, whereas there should be at least 20 female animals with implantation sites at necropsy. It is noted that since groups with fewer than 16 animals with implantation sites may be inappropriate.
- Sex ratio is not provided in the study report.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rabbit

Strain:

Chinchilla

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: KFM, Klein tier farm Madoerin AG, CH 4414 Fuellinsdorf / Switzerland
- Age at study initiation: Between 4 and 6 months
- Weight at study initiation: 2278 - 3334 grams (post coitum)
- Fasting period before study: no
- Housing: The rabbits mere housed individually in stainless steel cages equipped ujith automatic cleaning system
- Diet (ad libitum): Pelleted standard Kliba 341, rabbit maintenance diet (Klingentalmuehle AG, CH 4303 Kaiseraugst/Switzerland)
- Water (ad libitum): Tap water
- Acclimation period: 7 days (minimum) under test conditions, after veterinary examination.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 40 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: approx. 30-04-1986 To: 23-06-1986

Route of administration:

oral: gavage

Vehicle:

other: Distilled water with 0.5 % Cremophor EL

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test article mas weighed into a glass beaker on a tared precision balance (Mettler PK 300) and the vehicle added (w/v) . The mixtures were prepared using a homogenizer. During the daily administration period, homogeneity was maintained using a magnetic stirrer. The test article/vehicle mixtures were prepared daily prior to administration.

VEHICLE
- Amount of vehicle: 4 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration and homogeneity of the test article/vehicle mixtures were determined prior the first test article administration. During the treatment period, additional samples for confirmation of concentration, homogeneity and stability were taken once. Samples were taken immediately after preparation.
The concentration of the test item in triplicate samples taken for determination of homogeneity before the study was found to vary considerably (14-140% of the nominal concentration), with mean concentrations of 71 ± 8.3, 84 ± 64, and 70 ± 16% of the nominal concentration at doses of 0.1, 0.5, and 2.5 mg/kg bw/day, respectively. At the next sampling 10 days later, the mean concentrations were all within 90% of the nominal concentration, although the variability was still high (92 ± 37, 97 ± 45, and 99 ± 41% of the nominal concentration at the three doses, respectively).

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused: After acclimation the females mere housed with males until mating has been observed.
- M/F ratio per cage: 1/1, after mating individual housing
- Proof of pregnancy: not specified, the day after mating was recorded as Day 0 post coitum

Duration of treatment / exposure:

from Day 6 - Day 18 post coitum

Frequency of treatment:

Once daily

Duration of test:

28 days

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

vehicle control

Dose / conc.:

0.1 mg/kg bw/day (nominal)

Dose / conc.:

0.5 mg/kg bw/day (nominal)

Dose / conc.:

2.5 mg/kg bw/day (nominal)

No. of animals per sex per dose:

16 mated femle animals per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dosages mere based upon the results of the dose-finding embryotoxicity (including teratogenicity) study in the rabbit.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily, minimum

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily, minimum

BODY WEIGHT: Yes
- Time schedule for examinations: Daily, from day 0 until day 28 post coitum

FOOD CONSUMPTION: Yes
- Time schedule for examinations: The data were recorded on days 6, 11, 15, 19, 24 and 28 post coitum

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day: 28 post coitum (dams were killed by cervical dislocation and fetuses removed by caesarean section)
- Organs examined:
Postmortem examinations, including gross macroscopic examination
of all internal organs, with emphasis upon the uterus, uterine contents, position of fetuses in the uterus and number of corpora lutea, were performed and the data recorded. The fetuses were removed from the uterus, weighed, examined for gross external abnormalities and prepared for internal examinations.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes

The uteri (and uterine contents) of all pregnant females mere weighed on the scheduled day of necropsy. The weights mere used to determine the corrected body meight gain.
The uteri of the females tuhich were found at necropsy to be not pregnant were placed in an aqueous solution of ammonium sulfide to accentuate possible hemorrhagic areas of implantation sites. All tissues and organs of the females mere discarded. The stained trunks of fetuses and the sections of the heads of fetuses mere preserved .

Blood sampling:

not examined

Fetal examinations:

The following examinations were performed individually:

1) All fetuses were dissected carefully, the body cavities (thorax, abdomen, pelvis) and the organs were investigated and any abnormal findings recorded. The sex of each fetus was noted and recorded.

2) The skin was removed and the crania of all fetuses were examined for ossification.

3) The heads of all fetuses were fixed in a solution of trichloroacetic acid and formaldehyde. The heads were crosssectioned and the cephalic viscera were examined. Descriptions of any abnormalities were recorded. After evaluation, the individual sections were preserved in a solution of ethyl alcohol and glycerine (one head per container).

4) The trunks of all fetuses were placed in a solution of potassium hydroxide for clearing and stained ujith alizarin red (modified technique). The skeletons mere examined and all abnormalities and variations were recorded. The specimens mere preserved individually in plastic bags.

Statistics:

The following statistical methods mere used to analyze body weights, food consumption and reproduction data:
Univariate one-may analysis of variance uias used to assess the significance of intergroup differences if the variables could be assumed to follow a normal distribution. The Dunnett many-one t-test, based on a pooled variance estimate, urns used for intergroup comparisons (i.e. single treatment groups against the control group).
A one-may univariate analysis of variance based on Wilcoxon ranks together tuith the Kruskall-Wallis test was applied to the reproduction data parameters.
Fisher's exact test for 2x2 tables mas applied if the variables could be dichotomized without loss of information.
Individual values, means, standard deviations and t-statistics were rounded off before printing.

References:
1. C. W. Dunnett, A Multiple Comparison Procedure for Comparing several Treatments with a Control, J. Amer. Statist. Assoc. 50, 1096-121 (1955).
2. Rupert G. Miller, Simultaneous Statistical Inference, Springer Verlag, New York (1981).
3. Fisher, R.A. Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh <1950).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Group 4 (2.5 mg/kg bw): Salivation and dyspnea mere the symptoms mostly observed. Additionally, in two of the four females which died, ataxia mas noted, and in one female diarrhea mas observed. Salivation appeared for the first time on day 7 post coitum (second day of treatment) and at the last time on day 18 post coitum (last treatment-day). The signs and symptoms appeared about 30 to 60 minutes after test article administration and lasted up to 4 hours maximum.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Group 4 (2.5 mg/kg bw): Four females died prior caesarean section on day 28 post coitum. These four females were pregnant, also. In these females, eight, five, seven and eight embryos mere noted, respectively.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In group 4 (2.5 mg/kg bw): the mean body weight gain of dams during
the treatment period was clearly reduced in comparison to that of
group 1 (vehicle control).
Although statistically insignificant, this reduction was considered to be test article-related as the consequence of the distinct statistically significant reduction of the mean food consumption during the treatment period.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

In group 4 (2.5 mg/kg bw): the mean food consumption was distinct reduced (29.4 %) during the treatment period. This reduction was statistically significant and considered to be a test article-related effect.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The following incidental findings mere noted in groups 1 (vehicle control) and 3 (0.5 mg/kg bw): Indentations and areas mith grey-whitish discoloration on the surface of the kidneys mere noted in one female of group 1 and in one female of group 3.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

In group 4 (2.5 mg/kg bw): increase in preimplantation loss at 2.5 mg/kg bw/day (10.9 vs. 3.3 % of corpora lutea)
In general, in a teratogenicity study, pre-implantation loss is not related to treatment, because implantation occurs before treatment starts. Since the days of gestation were counted from mating and not corrected with one day, treatment might have influenced pre-implantation loss.

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

Other effects:

no effects observed

Details on maternal toxic effects:

No test article-related or statistically significant differences in the mean number of implantations, pups or embryonic deaths mere noted in the dose groups when compared to that of the vehicle control group. The differences evident tuere considered to be incidental and within the normal range of deviations for animals of this strain and age. The mean distribution of fetuses and resorptions within the uterine horns was similar in all groups.

Key result

Dose descriptor:

NOAEL

Effect level:

0.5 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food efficiency

mortality

pre and post implantation loss

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

not examined

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

A number of skeletal changes unrelated to treatment were seen in foetuses at all doses.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One foetus at the high dose had encephalocele with reduced brain size.

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

In foetuses, no effect that could be attributed to treatment was seen in sex ratio, body weight, external or internal malformations, skeletal abnormalities, or development.

Key result

Dose descriptor:

NOAEL

Effect level:

2.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

2.5 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

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

Dose response relationship:

yes

Conclusions:

The NOAEL for maternal toxicity was 0.5 mg/kg bw/day, based on mortality, clinical signs, decreased body weight and food intake at 2.5 mg/kg bw/day. The NOAEL for developmental toxicity is 0.5 mg/kg bw/day, based on the increase in pre-implantation loss at 2.5 mg/kg bw/day. The test substance was not teratogenic under the conditions of this study.

Executive summary:

The test substance was administered to groups of 16 single-mated female Chinchilla rabbits at dose levels of 0, 0.1, 0.5 and 2.5 mg/kg bw/day by gavage from day 6 to day 18 post coitum. The dose volume was 4 mL/kg bw. On day 28 all dams were sacrificed and their fetuses examined. Cholinesterase activity was not assessed.

Dams: At 2.5 mg/kg bw/day 4 dams died. Treatment-related signs of toxicity (ataxia, salivation, dyspnoea and diarrhoea) and reduced body weight gain were observed in the high dose group animals only.

Development: There were no differences between the treatment groups and the control group with regard to the mean numbers of implantations, corpora lutea, live or dead fetuses and resorptions. No effects on sex ratios of the live fetuses were seen. No changes in body weight, external or internal malformations, skeletal abnormalities, or development were noted. One fetus at 2.5 mg/kg bw/day had encephalocele with reduced brain size. This finding was not related to treatment.

NOAEL maternal: 0.5 mg/kg bw/day based on on reduced body weight gain and mortality at 2.5 mg/kg bw/day. NOAEL developmental: 2.5 mg/kg bw/day (highest dose tested). The test substance was not teratogenic in rabbits.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November - December 1988

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 (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:CD-BR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage, MI
- Age at study initiation: males: older than 10 weeks, younger than 1 year; older than 10 weeks, younger than 18 weeks
- Weight at study initiation: male: more than 250 grams; female: more than 180 grams
- Fasting period before study: no
- Housing: Stainless steel suspended cages in the same climate controlled room
- Diet (ad libitum): Ground Purina Certified Rodent Chow #5002
- Water (ad libitum): yes
- Acclimation period: Minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1988-11-21 To: 1988-12-16

Route of administration:

oral: gavage

Vehicle:

other: 2% aqueous Emulphor vehicle

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Following HPLC analysis, the dosing solutions were stored in a refrigerator at no greater than 5°C. On each day of test article administration, the stock solutions were removed from the refrigerator, stirred, and aliquots removed. The aliquots were also stirred during the time of test article administration.

VEHICLE
- Concentration in vehicle: 0.0025, 0.0085, or 0.03% solution
- Amount of vehicle: 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Remarks:

stability testing and analysis of the dosing solutions performed

Details on analytical verification of doses or concentrations:

Stability testing demonstrated that test article solutions in concentrations ranging from 0.0025% to 0.03% in 2% aqueous Emulphor vehicle remained stable for at least 4 weeks under conditions mimicking those used in the study.

Details on mating procedure:

- Impregnation procedure: natural insemination, employed over 9 days
- If cohoused: maximum of 2 female rats were housed overnight with a breeder male. On the following morning all males were removed and returned to individual cages
- M/F ratio per cage: 1/2
- Length of cohabitation: 9 days
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal smear; the day spermatozoa were identified microscopically was designated Day 0 of gestation.

Duration of treatment / exposure:

from Days 6 through 15 of gestation (during the stages of embryogenesis and early fetation)

Frequency of treatment:

total of 10 consecutive doses

Duration of test:

approx. 40 days

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

0.25 mg/kg bw/day (nominal)

Dose / conc.:

0.85 mg/kg bw/day (nominal)

Dose / conc.:

3 mg/kg bw/day (nominal)

No. of animals per sex per dose:

33 dams (28/group terminated on Day 20; 5/group terminated on Day 16).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses were based on a range-finding study where 5 pregnant rats/dose were dosed at days 6-15 of gestation with 0.25, 1.25, 2.0, 2.5, 3.25, 3.75, 5.5 or 7.5 mg/kg bw/day. All animals at ≥ 3.75 mg/kg bw/day died after first treatment. Effects on body weight and clinical signs were observed at doses of 2.0 mg/kg bw/day and higher.
- Time of day for dam sampling: Day 16 of gestation (for the purpose of obtaining blood and brain tissue for measurement of erythrocyte, plasma, and brain cholinesterase activity)
- Day of termination: On Day 20 of gestation (approximately 5 days after the last dose), the remaining dams were sacrificed by CO2 asphyxiation.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes, twice daily

DETAILED CLINICAL OBSERVATIONS: Yes, daily

BODY WEIGHT: Yes
- Time schedule for examinations: Day 0, 6, 8, 10, 12, 15, and 20 of gestation

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: brain, uterine content

CLINICAL PATHOLOGY: Yes
RBC, plasma, and brain cholinesterase activity measured for all dams terminated on Day 16 of gestation and for the first 20 of 28 pregnant dams/group terminated on
Day 20 of gestation

ORGAN WEIGHTS: Yes
1. Uterus: Intact weight determined on Day 20 of gestation for all dams
2. Placenta: Each placenta weighed individually on Day 20 of gestation for all dams

HISTOPATHOLOGY: When deemed necessary

OTHER:
Abortions: Observation of conceptus in the cage pan (i.e., whole or partial fetuses or placentas will be sufficient indication that the dam has aborted. Such dams will be terminated and necropsied. The number of corpora lutea and implantation sites will be recorded. Any remaining fetuses will be assessed for general development, sexed (if possible), and placed in 70 % ethanol for skeletal processing.

Ovaries and uterine content:

For all dams terminated on Day 20, the abdomen was opened, ovaries excised, and the corpora lutea graviditatis counted and recorded. The uterine horns were transected at the cervix, trimmed along the antimesometrial margin, removed, and weighed. Each uterine horn was opened longitudinally along the antimesometrial surface and the amniotic sacs displaced to one side to facilitate inspection of the uterine walls for the presence of resorptions.

Blood sampling:

Blood and brain tissue were collected from the first 20 of 28 pregnant dams/group (20/31 high-dose dams) and the methods outlined above for measuring cholinesterase were employed.

Fetal examinations:

Each fetus was removed from its amniotic membranes, the umbilical cord was severed close to its attachment to the fetus, and viability of the fetus was determined. Each fetus was blotted dry, removing blood and amniotic fluid, sexed, and weighed. Individual placentas corresponding to each fetus were trimmed of extraneous tissue, blotted dry, fixed and weighed.

- External examinations: Yes: all per litter

- Soft tissue examinations: Yes: half per litter

- Skeletal examinations: Yes: half per litter

- Head examinations: Yes: 20 fetuses per dose group (from 20 different litters where possible). Fetal brain cholinesterase determinations were made on representative fetusesfrom each treatment group. The fetuses selected for these determinations were the first fetus randomly selected for gross visceral examination from each litter.

Statistics:

Statistical analysis of the data consisted of application of one or more of the following tests: Dunn (1964), Dunnett's (1955; 1964), Fisher's exact (Pagano-HaIvorsen (1981), Healy (1972), and Kruskal-Wallis (1952).

Indices:

Reproductive Parameters:
1. Dams (Day 20 Termination):
a. Fertility index; ratio of number of pregnant animals/number of animals inseminated
b. Gestation index; ratio of number of females with live progeny/number of pregnant females
c. Litter size
d. Number of corpora lutea
e. Total number of implantations
1) Number of viable fetuses
2) Number of non-viable fetuses
3) Number of resorptions
f. Sex distribution (fetal)
g. Pre-implantation loss; ratio of corpora lutea minus total implants/number of corpora lutea
h. Post-implantation loss; ratio of total implantations minus number of viable progeny/number of implantations

Historical control data:

for Charles River Crl:CD BR Rat

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

High-dose group: tremors (transient), salivation, lacrimation, and wet (urine) soiling of the ventral surface, hypoactivity, chromodacryorrhea and convulsions

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

High-dose group: 6 treatment-related deaths

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

High-dose group: statistically significant (p<0.01) reduction in average body weight gain (38%) during the treatment period and a statistically significant (p<=0.05) reduction in both average weight gain during gestation and actual weight gain for the high-dose group. Body weight gain from the end of treatment (measured on Day 15) to the end of the study (measured on Day 20) was comparable between the high-dose and control groups indicating recovery.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

High-dose group: Corresponding to the reduction in body weight there was a statistically significant (p<=0.05) reduction in food consumption (10-17%) when assessed on both Days 12 and 16 of gestation. As with body weight, there was comparable food consumption between the high-dose and control groups by the end of the study.

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):

Plasma ChE activities were >20% reduced in dams at 3 mg/kg bw/day on day 16 of gestation. At day 16, erythrocyte ChE activities were >20% reduced in dams at 0.85 mg/kg bw/day and higher. By day 20, the plasma ChE activity had returned to normal, while that of erythrocyte ChE was still significantly reduced at 3 mg/kg bw/day. Brain ChE activity was reduced non-significantly and not in a dose related manner.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

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

Other effects:

no effects observed

Details on maternal toxic effects:

The test item produced no adverse effect on any measured maternal reproductive parameter. There were no statistically significant or toxicologically meaningful differences in pregnancy rates, number of does with viable progeny, litter size, numbers of corpora lutea, implantations, resorptions, or pre- and/or post-implantation loss when comparisons were made between the treatment groups and the control.

Key result

Dose descriptor:

NOAEL

Effect level:

0.25 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

clinical biochemistry

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

Anogenital distance of all rodent fetuses:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were several non-treatment-related external and/or visceral changes observed in each treatment group and the control group. Included among these changes were two major malformations. In one control group fetus, there was a rhinocephalic with cyclopia, displaced pinnae, agnathia, and astomia. One mid-dose fetus had exencephaly and possessed an omphalocele and supernumeraryappendages (1 additional pair of anterior and posterior appendages). These malformations were considered spontaneous in origin. All other external and visceral findings were also considered spontaneous in nature. None of the other findings occurred in a dose-dependent fashion and all are commonly observed in this strain of rats.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

There was no dose-dependent increase in any specific skeletal malformation, no remarkable delay in ossification of any skeletal element nor any apparent increase in the incidence of common skeletal variations (e.g., supernumerary ribs, additional pre-sacral vertebrae) in any test article group when comparisons were made with the control group.
Variations of the hyoid body (26% for the high-dose group) was the only noteworthy skeletal parameter found to be significantly different from control and this value was within this laboratory's historical control range (2-32%).

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

see "External malformations"

Other effects:

no effects observed

Description (incidence and severity):

There was no statistically significant increases in the litter or fetal incidence of malformations or selected variations when comparisons were made between the treatment groups and the control. In addition, no value fell outside of this laboratory's historical control range.

Key result

Dose descriptor:

NOAEL

Effect level:

0.85 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: increase in hyoid body variations at 3 mg/kg bw/day

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

other: mandibula

Key result

Developmental effects observed:

no

Lowest effective dose / conc.:

3 mg/kg bw/day (nominal)

Treatment related:

no

Conclusions:

The NOAEL for maternal toxicity was 0.25 mg/kg bw/day, based on decreased erythrocyte ChE activity on day 16 at 0.85 mg/kg bw/day. The NOAEL for developmental toxicity was 0.85 mg/kg bw/day, based on the increase in hyoid body variations at 3 mg/kg bw/day. The test substance was not teratogenic under the conditions of this study.

Executive summary:

The test item was administered orally to groups of 33 mated female Crl:CD-BR rats at concentrations of 0, 0.25, 0.85 and 3 mg/kg bw/day by gavage on days 6 to 15 of gestation. Parameters evaluated on the dams included: cholinestease determinations, body weights, food consumption, pregnancy rates, dams with live progeny, corpora lutea, implanations, resorptions, litter size, fetal weights and viability, placental weights, fetal sex ratio, pre- and post-implantation loss.

Dams: Tremor occurred in all dams at 3 mg/kg bw/day and six animals died at this dose level. There was a reduction in body weight gain and food consumption at 3 mg/kg bw/day.

Development: No effects on the developmental parameters were established in any dose group. Fetal weights were unchanged in comparison to the control group. The external and visceral examinations of the fetuses showed no treatment-related retardations, variations or malformations.

Cholinesterase (ChE) measurements: Plasma and ery ChE activities were lower in dams at 3 mg/kg bw/day on day 16 of gestation. By day 20, plasma activity had returned to normal, while ery ChE was still lower. In comparison to the control group brain ChE activity was lower at 0.85 and 3 mg/kg bw/day (28 % and 12 %, respectively) but as there was no dose-correlation, this finding was not considered to be related to the treatment. In the fetuses, brain ChE activity was unaffected; plasma and ery ChE could not be measured.

The NOAEL for maternal toxicity was 0.25 mg/kg bw/day, based on decreased erythrocyte ChE activity on day 16 at 0.85 mg/kg bw/day. The NOAEL for developmental toxicity was 0.85 mg/kg bw/day, based on the increase in hyoid body variations at 3 mg/kg bw/day. The test substance was not teratogenic under the conditions of this study.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

0.5 mg/kg bw/day

Species:

rabbit

Quality of whole database:

Klimisch 1

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

The information provided below was taken from the original dossier submitted in 2002 for inclusion of the test substance to Annex I of Directive 91/414/EEC and has been previously evaluated in the DAR and subject to peer review by EFSA and Member States.

 

Schlüter (1981): Groups of 25 female rats were mated overnight with untreated males (in a ratio of one male to two females) and were given the test substance in a 0.5% aqueous Cremophor emulsion at daily doses of 0, 0.3, 1, or 3 mg/kg bw/day by gavage on days 6-15 of gestation. The first day of gestation was that on which sperm was found in a smear obtained the morning after mating. Control females received the same volume (10 mL/kg bw) of the aqueous emulsion. On day 20 of gestation, the dams were narcotised with carbon dioxide and the foetuses were removed. Litter size, average foetus weight per litter, sex, external and visceral abnormalities, and skeletal malformations and development were noted. Cholinesterase activity was not measured. Eighteen dams receiving 3 mg/kg bw/day showed signs of toxicity (trembling and recumbency), and two died; however, the time of death was not given and the cause of death could not be established. A 15% reduction in average weight gain was seen during treatment among dams receiving 3 mg/kg bw/day in comparison with controls. The average placental weight of animals at the high dose was significantly lower than that in controls (8%). Treatment did not affect litter size, number of resorptions, number of foetuses, average foetal weight, sex ratio, incidence of alterations in development, or the type or number of malformations. The most frequent manifestations were nodulations on ribs, which were found in two foetuses from one litter of a dam at 0.3 mg/kg bw/day and in four foetuses of three litters of dams at 1 mg/kg bw/day. The other malformations observed were general oedema, abdominal fissure, and anophthalmia in one foetus at 0.3 mg/kg bw/day. No malformations were observed in foetuses at the high dose. Based on clinical signs, decreased body weight gain, and decreased placenta weights at 3 mg/kg bw/day, the NOAEL for maternal toxicity is 1 mg/kg bw/day. The NOAEL for developmental toxicity is 3 mg/kg bw/day, based on the absence of developmental effects at the highest dose tested. The test item was not teratogenic under the conditions of this study. This study is considered acceptable as supplementary study to Clemens, 1989.

 

Clemens (1989): The investigation of the test item's potential teratogenic effects was conducted equivalent to OECD 414. Test item was administered orally to groups of 33 mated female Crl:CD-BR rats at concentrations of 0, 0.25, 0.85 and 3 mg/kg bw/day by gavage on days 6 to 15 of gestation. Parameters evaluated on the dams included: cholinestease determinations, body weights, food consumption, pregnancy rates, dams with live progeny, corpora lutea, implanations, resorptions, litter size, fetal weights and viability, placental weights, fetal sex ratio, pre- and post-implantation loss. Tremor occurred in all dams at 3 mg/kg bw/day and six animals died at this dose level. There was a reduction in body weight gain and food consumption at 3 mg/kg bw/day. No effects on the developmental parameters were established in any dose group. Fetal weights were unchanged in comparison to the control group. The external and visceral examinations of the fetuses showed no treatment-related retardations, variations or malformations. Plasma and ery ChE activities were lower in dams at 3 mg/kg bw/day on day 16 of gestation. By day 20, plasma activity had returned to normal, while ery ChE was still lower. In comparison to the control group brain ChE activity was lower at 0.85 and 3 mg/kg bw/day (28 % and 12 %, respectively) but as there was no dose-correlation, this finding was not considered to be related to the treatment. In the fetuses, brain ChE activity was unaffected; plasma and ery ChE could not be measured. The NOAEL for maternal toxicity was 0.25 mg/kg bw/day, based on decreased erythrocyte ChE activity on day 16 at 0.85 mg/kg bw/day. The NOAEL for developmental toxicity was 0.85 mg/kg bw/day, based on the increase in hyoid body variations at 3 mg/kg bw/day. The test substance was not teratogenic under the conditions of this study.

 

Mackenzie (1982): Groups of 20 double-mated female rabbits were given the test substance orally at doses of 0, 0.1, 0.3, or 1 mg/kg bw/day on days 6-18 of gestation. Day 0 was the day the rabbits mated. They were observed daily for clinical signs of toxicity and were weighed initially, periodically during the test, and at termination of the study. Once the pups had been removed, the ovaries and uteri of the dams were examined, and the foetuses were examined grossly and prepared for evaluations of soft tissues and the skeleton. The numbers of corpora lutea, implantations, resorptions, live and dead foetuses, and anomalies were also determined. Cholinesterase activity was not assessed. Dams given doses ≥ 0.3 mg/kg bw/day showed signs of toxicity, with decreased body-weight gain (34 and 49% for 0.3 and 1.0 mg/kg bw/day respectively), bloody nasal discharge, and white, mucoid ocular discharge. At 1.0 mg/kg bw/day decreased gravid uterine weight was observed (20%). Treatment did not affect the number of litters, number of pups per litter, pregnancy rate, number of corpora lutea, implantations, or gross abnormalities. A slight apparent increase in the foetal incidence of post-implantation losses is attributed to high uncertainty due to low number of foetuses (and litters) in the study. Mean foetal weight was slightly depressed at 1 mg/kg bw/day (7%). One dam at 0.3 mg/kg bw/day aborted one dead pup, and two dams at 1 mg/kg bw/day aborted eight dead pups and had seven late resorptions. In addition, one dead foetus was found in each of two litters at the high dose. The most common developmental variation observed was the left carotid arising from the innominate, which occurred at doses ≥ 0.1 mg/kg bw/day. A dose-related response was not observed. This anomaly was not seen in the controls and in only one of 31 litters (3.2%) of historical controls at the laboratory where the study was performed10. Based on the total set of information, this effect is not considered to be related to treatment. An increased incidence of accessory skull bones was also seen in all treated groups. A significant increase in the incidence of chain-fused sternebrae was seen at 1 mg/kg bw/day, and this anomaly was also seen at 0.3 mg/kg bw/day. These skeletal anomalies were found at maternal toxic doses only, and are probably due to the maternal toxicity. Two foetuses in one litter at the high dose had aortic arches with a common truncus, which was considered to be a major malformation. Other skeletal malformations, which occurred at doses ≥ 0.3 mg/kg bw/day, included fused ribs, scoliosis, absent vertebrae (thoracic, lumbar, sacral, and caudal), and bipartite or malformed centra. These anomalies occurred in only one or two foetuses in single litters. Furthermore, several of the anomalies were clustered within a single foetus, indicating that they were not likely to be related to treatment. The NOAEL for maternal toxicity was 0.1 mg/kg bw/day, based on decreased body weight gain and clinical signs (bloody nasal discharge, and white, mucoid ocular discharge) at 0.3 mg/kg bw/day. The doses of 0.3 and 1.0 mg/kg bw/day exceed the MTD. The NOAEL for developmental toxicity was 0.1 mg/kg bw/day, based on skeletal findings at 0.3 mg/kg bw/day (decreased foetal weight and skeletal findings were observed at 1.0 mg/kg bw/day). The test substance was not teratogenic under the conditions of this study.

Although there is no reference to a specific test guideline in the study report, the following deviations were noted from the revised OECD TG 414 (2001): The test substance was not administered until the day of scheduled kill, but only during the period of organogenesis. Although 20 dams/group were used in the study, less than 16 dams/group had implantation sites at necropsy. Food intake was not recorded. Further, cholinesterase activity was not measured. Due to study limitations, the study is acceptable to be considered together with the developmental toxicity study in rabbits by Becker, 1986.

 

Becker (1986): The investigation of the test item's potential teratogenic effects was conducted equivalent to OECD 414. The test substance was administered to groups of 16 single-mated female Chinchilla rabbits at dose levels of 0, 0.1, 0.5 and 2.5 mg/kg bw/day by gavage from day 6 to day 18 post coitum. The dose volume was 4 mL/kg bw. On day 28 all dams were sacrificed and their fetuses examined. Cholinesterase activity was not assessed. At 2.5 mg/kg bw/day 4 dams died. Treatment-related signs of toxicity (ataxia, salivation, dyspnoea and diarrhoea) and reduced body weight gain were observed in the high dose group animals only. There were no differences between the treatment groups and the control group with regard to the mean numbers of implantations, corpora lutea, live or dead fetuses and resorptions. No effects on sex ratios of the live fetuses were seen. No changes in body weight, external or internal malformations, skeletal abnormalities, or development were noted. One fetus at 2.5 mg/kg bw/day had encephalocele with reduced brain size. This finding was not related to treatment. NOAEL maternal: 0.5 mg/kg bw/day based on on reduced body weight gain and mortality at 2.5 mg/kg bw/day. NOAEL developmental: 2.5 mg/kg bw/day (highest dose tested). The test substance was not teratogenic in rabbits.

 

A summary of the results of developmental toxicity studies is presented in Table 2.

 

Table 2: Summary of developmental toxicity studies

Type of study	Species		NOAEL	LOAEL	Critical effects	Reference
			(mg/kg bw/day)	(mg/kg bw/day)
teratogenicity	rat	maternal	1.0	3.0	Clinical signs, decreased body weight gain, decreased placenta weight	Schlüter, 1981
		developmental	3.0	-	No effects at highest dose tested
teratogenicity	rat	maternal	0.25	0.85	Decreased erythrocyte ChE activity	Clemens, 1989
		developmental	0.85	3.0	Increase hyoid body variations
teratogenicity	rabbit	maternal	0.1	0.3	Decreased body weight gain, clinical signs	Mackenzie, 1982
		developmental	0.3	1.0	Decreased foetal weight, skeletal findings
teratogenicity	rabbit	maternal	0.5	2.5	Mortality, clinical signs, decreased bodyweight and food intake	Becker, 1986
		developmental	2.5	-	No effects at highest dose tested

Justification for classification or non-classification

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

The available studies are considered reliable for this assessment.

Based on the above stated assessment of the reproduction toxicity potential of the test substance, it is deemed that the compound is not toxic to the reproduction and do not need to be classified according to CLP (Regulation (EC) No 1272/2008 of the European Parliament and of the Council) as implementation of UN-GHS in the EU and as amended for the eighteenth time in Regulation (EU) 2022/692.