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Diss Factsheets

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997

Materials and methods

Test guideline
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.

Test material

Constituent 1
Chemical structure
Reference substance name:
Fenamiphos
EC Number:
244-848-1
EC Name:
Fenamiphos
Cas Number:
22224-92-6
Molecular formula:
C13H22NO3PS
IUPAC Name:
{ethoxy[3-methyl-4-(methylsulfanyl)phenoxy]phosphoryl}(propan-2-yl)amine
Test material form:
solid: crystalline

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Effect levels (P0)

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

Target system / organ toxicity (P0)

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

Results: P1 (second parental generation)

General toxicity (P1)

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 / performance (P1)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Effect levels (P1)

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

Results: F1 generation

General toxicity (F1)

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

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

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

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

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Effect levels (F2)

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

Target system / organ toxicity (F2)

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

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

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.