Pirimiphos-methyl

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

- Oral: NOAEL for systemic toxicity (based on the inhibition of plasma cholinesterase activity): 10 ppm (corresponding to 1 mg/kg bw/day); NOAEL for reproductive toxicity: 160 ppm (12 and 15 mg/kg bw/day for F0 and F1 respectively), highest tested dose; EPA 83 -4, Barton 1995

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Jun 1993 to 07 Mar 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: (F0) 6 weeks
- Weight at study initiation: (F0) Males and females: ca 100 g
- Housing: The F0 animals were initially housed 2 per cage in polypropylene cages with stainless steel grid bottoms, mesh tops and food hoppers. The cages measured 42 x 27 x 20 cm and were suspended on racks containing 6 rows of 4 cages. Males and females were racked separately. Excreta were collected on a tray lined with absorbent paper, suspended beneath each cage. Three days prior to the initiation of mating, the males were transferred to individual cages. For mating, the females were transferred to the cage of the appropriate co-group male. Mated females were transferred to individual solid bottomed cages of the same dimensions, in which sterilised white wood shavings were provided as bedding. White paper tissue was supplied to each mother for incorporation in the nest; this was replaced when it became soiled. Dams and their litters retained this type of cage until termination. F1 animals selected as parents fo the next generation were housed 2 per grid-bottom cage, sexes separate, and then followed the same caging regime as described for the F0 animals.
- Diet: Rat and Mouse Breeder Diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 2
- Humidity (%): 50 ± 15
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 18 Jun 1993 To: 7 Mar 1994

Route of administration:

oral: feed

Vehicle:

ethanol

Details on exposure:

DIET PREPARATION
Fresh batches of treated diets were prepared weekly during the study, and used within one week of preparation. An appropriate quantity of the test material was dissolved in a suitable volume of ethanol. This solution was added to a suitable quantity of untreated diet, then mixed for at least 2 h using a Hobart mixer with fan assisted venting to form a premix. A control premix was prepared using the same proportion of ethanol and untreated diet. The formulated diets for the Control, Intermediate and High doses were prepared by dilution of the appropriate premix with untreated diet to give the desired concentrations. The Low dose diet was prepared by dilution of the High dose diet with untreated diet. The formulations were mixed on a Winkworth tumblemixer for at least 20 min

Details on mating procedure:

- M/F ratio per cage: Animals were generally paired on a one male to one female basis, sibling matings being avoided. On one occasion the number of males in a group was reduced by mortality, and so one male was paired on a one male to 2 females basis, such that all females were paired.
- Length of cohabitation: until mating was detected or 7 nights had elapsed
- Proof of pregnancy: A vaginal lavage was examined early each morning, commencing on the morning of pairing, until a mating sign was detected; the day of detection of sperm in the lavage, or of a copulatory plug in situ was considered to be Day 0 of gestation
- After 7 days of unsuccessful pairing replacement of first male by another male with proven fertility; If no mating sign was detected during that time, a rest period of 2 days was allowed before the female was placed with a second co-group male, which had already mated. After 7 nights with the second male, if no mating sign had been detected, attempts to breed from the female ceased and she was transferred to an individual, solid bottom cage
- Further matings after two unsuccessful attempts: no
- Any other information: The stage of the oestrous cycle in each lavage was recorded to assist with interpretation of mating performance and to indicate possible adverse effects on the oestrus cycle

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On 7 occasions triplicate samples were taken from each formulated diet: comencement of treatment (week 1), F0 pre-mating period (week 2), F0 pre-mating period (week 7), F0 gestation period (week 12), F1 pre-mating period (week 19), F1 pre-mating period (week 26), F1 lactation period (week 34)

The method may be summarized as follows: Ten gram aliquots of treated diet were weighed into 250 mL glass centrifuge bottles. Toluene was added to the sample, which was allowed to soak for four hours, then homogenized using a high speed blender. Following centrifugation to separate the solid and liquid phases, an aliquot of the toluene supernatant was collected and dried over granular anhydrous sodium sulfate. A portion of the dried aliquot was placed into an autosampler vial for analysis using a gas chromatograph equipped with a flame photometric detector (FPD). The samples were chromatographed on a 15 meter, DB-1 column.

Duration of treatment / exposure:

Commencing at ca 6 weeks of age, F0 animals were treated for 10 weeks prior to mating for the production of F1 litters. Treatment continued throughout the mating, gestation and lactation periods, until termination after weaning of these litters. F1 animals were weaned onto the same diets as were fed to their respective parents. The selected F1 animals were treated for ca 12 weeks after weaning, prior to mating. Treatment then continued for both sexes throughout the mating, gestation and lactation periods, until termination after weaning of the F2 litters.

Frequency of treatment:

Continuous

Details on study schedule:

- F1 parental animals not mated until 9 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 12 weeks

F1 WEANING
For the selected pups, weaning occured on Day 24 of lactation, when they were removed from their mother and re-housed 2 per grid bottomed cage, sexes on seperate racks. The remaining F1 pups and all F2 pups remained with their mother until termination

Dose / conc.:

10 ppm

Remarks:

Low dose group - Equivalent to 1 mg/kg bw/day (F0 and F1 generations)

Dose / conc.:

40 ppm

Remarks:

Middle dose group - Equivalent to 3 and 4 mg/kg bw/day (F0 an F1 generations, respectively)

Dose / conc.:

160 ppm

Remarks:

High dose group - Equivalent to 12 and 15 mg/kg bw/day (F0 an F1 generations, respectively)

No. of animals per sex per dose:

- F0: 28
- F1: 24

Control animals:

yes, plain diet

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule: morning and evening every day

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: daily
- Clinical observations included behaviour, appearance, respiration, skin and coat condition.

BODY WEIGHT:
- Weights of F0 animals were recorded one week prior to the first day of test material administration, then weekly thereafter until the start of the mating period. Males continued weekly weighing until termination. Weights for females were also recorded on Day 0 of gestation (the day of detection of a positive mating sign) and Days 7, 14 and 20 of gestation, then on Days 1, 7, 14 and 21 of lactation (where the day of birth of the litter was designated Day 0 of lactation).
- Post-weaning F1, animals were weighed weekly from selection until the start of their mating period, after which they followed the same regime of weighing as described for F0 animals.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined: yes
- Time schedule: For F0 animals, food consumption was measured over the week prior to the first administration of test material and then weekly thereafter, until the start of the mating period. For males, consumption continued to be measured over complete one week periods, except during periods of cohabitation with females. For females, weekly food consumption measurements resumed over the 3 weeks of gestation (Days 0-7, 7-14, 14-20) and of lactation (Days 0-7, 7-14, 7-21). For F1 animals, measurement began shortly after selection and then followed the same regime as for the F0 animals.

OBSERVATIONS ON FEMALES DURING THE LACTATION PERIOD
- The females were allowed to litter normally. The day on which parturition commenced was designated Day 0 of lactation.
- The duration of gestation days was calculated.
- Any deficiencies in maternal care were recorded: points looked for included inadequate construction and cleaning of the nest, pups left scattered and cold, physical abuse of pups, or apparently inadequate lactation or feeding

CLINICAL CHEMISTRY
- Plasma and red blood cell cholinesterase activity were assessed

Oestrous cyclicity (parental animals):

The stage of the oestrous cycle in each vaginal lavage (during mating) was recorded to assist with interpretation of mating performance and to indicate possible adverse effects on the oestrus cycle.

Sperm parameters (parental animals):

Parameters examined in all male generations: From the High dose and Control groups, the testes and epididymides were examined for qualitative and quantitative effects on spermatogenesis (except for premature decedents).

Following a short (less than 24 h) fixation in bouin’s fluid, a single transversesection disc was cut from each testis. These discs were then transferred to absolute alcohol, followed by processing and embedding in paraffin wax. Sections were cut and stained with haematoxylin and PAS (periodic acid-Schiff), and examined by light microscopy.
For each pair of testes, the number of tubule cross-sections per mm2 was recorded and averaged from 5 microscope fields (each of 2.54 mm2), the calculation of the relative tubule length was performed according to the method of Lennox et al (1970), using the product of testis volume and the average number of tubule crosssections per mm2. From each pair of testes, 10 cross-sections of tubules in Stage VII of the cycle of the seminiferous epithelium, staged according to Leblond and Clermont (1952), were examined for the numbers of preleptotene spermatocytes,
pachytene spermatocytes, and Sertoli cells. The ratio of pachytene spermatocytes to Sertoli cells was calculated. The diameters of 20 cross-sections in Stage VII were also recorded using an eyepiece graticule (with 86 units equalling 1 mm). In cases where one of the testes was grossly abnormal, all assessments were made from the unaffected organs.
After an initial preservation in Hams medium, an Eosin-stained smear of sperm from one cauda epididymis from each male was prepared and then examined for morphological abnormalities using criteria described by Wyrobek and Bruce (1975)

One thousand sperm were evaluated per animal for the following abnormalities:
A: Sperm hood upturned or elongated
B: Banana-shaped sperm head
C: Amorphous sperm head
D: Sperm tail tightly coiled
E: Sperm tail acutely folded

Litter observations:

PARAMETERS EXAMINED
The number of live pups born and the number found dead in each litter was recorded as soon as possible after completion of parturition. The live pups were sexed, counted, examined for the presence of milk in the stomach and for any externally visible abnormalities daily up to Day 4 of lactation; they were counted and examined for abnormalities again on Days 7, 14 and 21. Pre-weaning F1 and F2 pups were weighed en masse by litter (sexes separate) on Days 1, 4, 7 and 14 of lactation. The pups were weighed individually on Day 21 of lactation, and the total litter weight was also recorded. Where practical, any pups found dead or killed during lactation were sexed and examined as above.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
Males were killed around the time of weaning of the first litters. Females were killed at or shortly after the time of weaning of their litter.

GROSS NECROPSY
All F0 and F1 animals were subjected to a necropsy, consisting of an external examination, followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities. Any gross lesions were described and representative samples taken and fixed in neutral buffered 10 % formalin.

ORGAN WEIGHTS / FIXATION
- The following organs were weighed (where indicated) and fixed: ovaries, uterus, cervix and vagina, testes (weight and volume recorded individually), epididymides (weighed individually), seminal vesicles, coagulating gland (weighed), prostate gland (weighed), pituitary gland, adrenal glands, brain (weighed).
- The female reproductive tract was examined for signs of pregnancy and the number of visible implantation sites was recorded.
- The testes were fixed in Bouin’s fluid, and one cauda epididymis was preserved in Ham’s F10 medium. The remaining tissues and organs were fixed in 10% formalin.
- Carcasses were discarded following these procedures.
- 0.1 g sample of brain (from the frontal cortex) was removed, snap frozen in liquid nitrogen and stored at -20°C until analysed.

HISTOPATHOLOGY
- From the High dose and Control groups, the testes and epididymides were examined for qualitative and quantitative effects on spermatogenesis
- Following a short fixation in bouin’s fluid, a single transverse sections was cut from each testis. These discs where then transferred to absolute alcohol, followed by processing and embedding in paraffin wax. Section were cut and stained with haematoxylin and PAS and examined by light microscopy.
- After an initial preservation in Hams medium, an Eosin-stained smear of sperm form one cauda epididymis form each male was prepared and then examined for morphological abnormalities.
- One thousand sperm were evaluated per animal for abnormalities
- The remaining epididymis, together with the seminal vesicles, coagulating gland, prostate gland and pituitary gland form each male, and the ovaries, uterus, cervix, vagina and pituitary gland from male were processed, and a haematoxylin and eosin stained section from each organ was examined histologically
- Brain cholinesterase (Brain ChE) was measured in the sample of the brain

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were subjected to postmortem examinations (macroscopic and/or microscopic examination)
- Pre-weaning: offspring found dead or killed before Day 14 of lactation were sexed, examined for externally visible abnormalities and for the presence of milk in the stomach. Offspring dying on or after Day 14 were subjected to a gross necropsy
- At Weaning: From each litter, 2 male and 2 female pups were necropsied. The remaining pups (except F1 weanlings selected for rearing to produce the next generation) were killed after external examination and the carcasses discarded without necropsy.

GROSS NECROPSY
- Pre-weaning: the cranial, thoracic and abdominal contents were examined macroscopically; any findings were recorded and abnormal tissues sampled, as appropriate.
- At weaning: The necropsy consisted of an external examination, followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities. Any gross lesions were described and representative samples taken and fixed in neutral buffered 10% formalin. Carcasses were discarded following these procedures. The remaining pups were killed after external examination and the carcasses discarded without necropsy.

Statistics:

Where considered appropriate to assist with interpretation, statistical analyses were applied to determine the statistical significance of differences from Control. Cholinesterase data were generally analysed by analysis of variance, with pairwise comparisons made using Fisher’s F-protected t-test (Snedecor and Cochran, 1980). Where necessary, a log transformation was used to stabilise the variances. Where a log transformation failed to stabilise the variances, the Kruskal-Wallis test, followed by Dunn’s procedure was used (Hollander and Wolfe, 1973).
Organ weight data were analysed by analysis of variance and by analysis of covariance (Snedecor and Cochran, 1980) using the terminal body weight as the single covariate. Treatment means were compared using an F-protected Least Significant Difference procedure.

Reproductive indices:

- Fertility index = Number of pregnant females or siring males/Number Paired
- Gestation index = Number bearing live pups/Number pregnant

Offspring viability indices:

- Birth index = Total number of pups born (live and dead) / Number of implantation scars
- Live birth index = Number of pups live on Day 0 of lactation / Total number born
- Viability index = Number of pups live on Day 4 of lactation / Number live on Day 0
- Lactation index = Number of pups live on Day 21 of lactation / Number live on Day 4
- Overal survival index = Number of pups live on Day 21 of lactation / Total number of pups born (live and dead)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

None of the observed clinical observations were considered to be associated with treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Among males, body weight performance was similar in all groups.
Among females, mean weight gain at 160 p.p.m. during the premating and gestation periods were marginally lower than Control; slight differences in body weight performance during lactation were not obviously associated with treatment. Body weight performance of females at 10 and 40 p.p.m. was essentially similar to Control.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Slight intergroup differences in food consumption were considered too small to be attributed to treatment.

Food efficiency:

no effects observed

Description (incidence and severity):

At all times, the achieved dosages were proportional to the nominal dietary concentrations.

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):

Mean Plasma cholinesterase:
At 40 and 160 ppm, mean plasma cholinesterase activity during treatment was consistently and significantly lower than Control (38-86 % and 16-56 % of Control for 40 and 160 ppm, respectively). At 10 ppm, mean plasma cholinesterase activity in the F0 generation was significantly lower than Control (61-78% of Control).

Red Blood Cell Cholinesterase:
At 160 ppm, mean red blood cell cholinesterase activity during treatment was generally significantly lower than Control (generally 52-67% of Control). At 40 ppm, there was some indication of reduction in red blood cell cholinesterase activity, especially among the F0 generation. At 10 ppm, there were no consistent differences in red blood cell cholinesterase activity compared with Control.

Brain Cholinesterase:
At 160 ppm, mean brain cholinesterase activity was lower than Control (47-87 % of Control), with the differences attaining statistical significance. At 40 ppm, brain cholinesterase activity of females was slightly lower than Control (47-56 % of Control), but with the differences attaining statistical significance. At 10 ppm, and for males at 40 ppm, brain cholinesterase levels were essentially similar to Control.

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:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

The histological assessments did not indicate any obvious adverse effect of treatment.

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

Description (incidence and severity):

Mating performance, fertility of both sexes and the duration of gestation were similar in all groups.
The mean number of implants were essentially similar in all groups.

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

10 ppm

Based on:

test mat.

Remarks:

corresponding to 1 mg/kg bw/day

Sex:

male/female

Basis for effect level:

clinical biochemistry

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity

Effect level:

160 ppm

Based on:

test mat.

Remarks:

corresponding to 12 mg/kg bw/day

Sex:

male/female

Basis for effect level:

reproductive function (sperm measures)

reproductive performance

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

None of the observed clinical observations were considered to be associated with treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Among males, body weight performance was similar in all groups.
Among females, mean weight gain at 160 p.p.m. during the premating and gestation periods were marginally lower than Control; slight differences in body weight performance during lactation were not obviously associated with treatment. Body weight performance of females at 10 and 40 p.p.m. was essentially similar to Control.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Slight intergroup differences in food consumption were considered too small to be attributed to treatment.

Food efficiency:

no effects observed

Description (incidence and severity):

At all times, the achieved dosages were proportional to the nominal dietary concentrations.

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Plasma Cholinesterase:
At 40 and 160 ppm, mean plasma cholinesterase activity during treatment was consistently and significantly lower than Control (38-86 % and 16-56 % of Control for 40 and 160 ppm, respectively). At 10 ppm, mean plasma cholinesterase activity for F1 animals, activity was generally lower than Control except for F1 males sampled shortly prior to mating, although only the value for F1 females at termination attained statistical significance.

Red Blood Cell Cholinesterase:
At 160 ppm, mean red blood cell cholinesterase activity during treatment was generally significantly lower than Control (generally 52-67% of Control), although the value for F1 females at termination was similar to Control (93% of Control). At 40 ppm, there was some indication of reduction in red blood cell cholinesterase activity. At 10 ppm, there were no consistent differences in red blood cell cholinesterase activity compared with Control.

Brain Cholinesterase:
At 160 ppm, mean brain cholinesterase activity was lower than Control (47-87 % of Control), with the differences attaining statistical significance except for F1 males. At 40 ppm, brain cholinesterase activity of females was slightly lower than Control (47-56 % of Control), but with the differences attaining statistical significance. At 10 ppm, and for males at 40 ppm, brain cholinesterase levels were essentially similar to Control.

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:

no effects observed

Description (incidence and severity):

There were no statistically significant intergroup differences in the weights of any of the organs assessed.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

The histological assessments did not indicate any obvious adverse effect of treatment.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

Testicular tubular atrophy was seen in 6 F1 males at 160 ppm, together with one Control F1 male. In 4 of these males at 160 ppm, the finding was only very mild, and in the 2 males with moderate atrophy the lesion was only unilateral. Furthermore, only one of the males with moderate atrophy, together with one male with very mild atrophy and a further male with no atrophy, failed to sire a litter. Also, the morphometic measurements and quantitative assessment of cell types for animals with tubular atrophy were not obviously different from those animals without atrophy. These parameters of the detailed examination of the testes were similar in the Control and High dose groups. The incidence of sperm abnormalities was similar in all groups.

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating performance, fertility of both sexes and the duration of gestation were similar in all groups.
The mean number of implants were essentially similar in all groups.

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

10 ppm

Based on:

test mat.

Remarks:

corresponding to 1 mg/kg bw/day

Sex:

male/female

Basis for effect level:

clinical biochemistry

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity

Effect level:

160 ppm

Based on:

test mat.

Remarks:

corresponding to 15 mg/kg bw/day

Sex:

male/female

Basis for effect level:

reproductive function (sperm measures)

reproductive performance

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

The values for Birth, Live Birth, Viability, Lactation and Overall Survival Indices at all dose levels in both generations were similar to the corresponding Control values.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Slight intergroup differences in litter and pup weights did not appear to indicate any adverse effect of treatment.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

One pup from the Control Litter was killed in early lactation with absent tail, bladder distended and apparent ataxia. One pup from the 10 ppm Litter had a pale area on kidney. One pup from 160 ppm Litter had a bipartite spleen. None of these abnormalities were considered to have been associated with treatment.

Histopathological findings:

no effects observed

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

160 ppm

Based on:

test mat.

Remarks:

corresponding to 12 mg/kg bw/day

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

The values for Live Birth, Viability, Lactation and Overall Survival Indices at all dose levels in both generations were similar to the corresponding Control values.
At 160 ppm the mean number of F2 pups born was slighty lower than the Control value, with consquent slight lowering of the Birth Index.
The mean number of F2 pups on Day 21 of lactation at 160 ppm was greated than the Control value, and it was therefore considered that the slightly lower Birth index as of doubtful biological importance.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Slight intergroup differences in litter and pup weights did not appear to indicate any adverse effect of treatment.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

One pup from the 10 ppm Litter had one eye absent. One pup from the Control Litter had a pale area on kidney. One pup from the 40 ppm Litter had left eye opaque and stomach distended. One pup from the Control Litter had free blood in cranial cavity. None of these abnormalities were considered to have been associated with treatment.

Histopathological findings:

no effects observed

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

160 ppm

Based on:

test mat.

Remarks:

corresponding to 15 mg/kw bw/day

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Table 1. Analysis of formulated diets: means and overall means of corrected percent nominal for test substance treated diets

Study week	Mean corrected percent nominal
	10 ppm	40 ppm	160 ppm
1	77.8	87.1	85.8
2	90.1	81	81.3
7	79.6	83.1	83.4
12	96.8	87.1	81.7
19	93.8	89.3	87
34	78.4	74.5	77.6
Overall means	86.1	83.7	82.8

Values represent analyses of samples from the top, middle and bottom sections of bulk feed batch

Table 2. Cholinesterase activities (mean; absolute and % of control) in Sprague Dawley rats (14/group) fed diets containing the test substance over 2 generations

	F0 to F1 generation			F1 to F2 generation
Timing	pre-dosing	pre-mating	sacrifice	pre-mating	sacrifice
Dose (ppm)
Erythrocyte
Male       0^#	989	1250	1476	945	1272
10 (%)	123	96	90	79	92
40 (%)	102	78*	83**	99	83**
160 (%)	144	64**	67***	64	63**
Females    0^#	1575	1513	1234	1085	439
10 (%)	113	89*	94	108	112^+
40 (%)	110	79***	73***	83	97^+
160 (%)	105	54***	52***	53**	93^+
Brain
Males       0^£	N.P.	N.P.	16113	N.P.	12558
10 (%)	N.P.	N.P.	96	N.P.	103
40 (%)	N.P.	N.P.	96	N.P.	96
160 (%)	N.P.	N.P.	87***	N.P.	82
Females    0^£	N.P.	N.P.	14735	N.P.	14674
10 (%)	N.P.	N.P.	90*^$	N.P.	94
40 (%)	N.P.	N.P.	74***	N.P.	84*
160 (%)	N.P.	N.P.	47***	N.P.	56***

N.P. - not performed    ^$- only 2/14 animals below control range        

⁺ - Control value low  ^£- iU/mg          ^#- iU/l

* P<0.05     ** P<0.01        *** P<0.001

 

 

Table 3. Reproductive Performance for F0 and F1 and Litter parameters for F1 and F2

Observation	Dose Group (mg/kg/day)
	0	10		40	160
F0 Reproductive Performance
Mean precoital interval (days)	2	2		2	2
Males
Males placed with females	28	28		28	28
Mated	27	27		26	27
Male mating index	96	96		93	96
Males with females pregnant	26	25		25	24
Male fertility index	93	89		89	86
Females
Females placed with males	28	28		28	28
Number inseminated	27	25		25	24
Female mating index	96	89		89	86
Pregnant	27	25		26	25
Female fertility index	96	93		100	93
Mean gestation interval (days)	22.0	22.1		22.0	22.2
Number of litters	27	25		25	23
Gestation index	100	100		100	96
F1 Reproductive Performance
Mean precoital interval (days)	3	2		2	2
Males
Males placed with females	23	23		24	24
Mated	19	23		23	23
Male mating index	83	100		96	96
Males with females pregnant	21	21		23	21
Male fertility index	91	91		96	88
Females
Females placed with males	23	24		24	24
Number inseminated	21	22		23	22
Female mating index	91	92		96	92
Pregnant	21	22		23	22
Female fertility index	91	92		96	92
Mean gestation interval (days)	21.9	21.7		21.7	21.9
Number of litters	21	22		23	21
Gestation index	100	100		100	95
Litter Parameters
F1 Generation
Mean Implantation Sites	17.3		16.0	16.5		16.1
Number born live	417		369	375		339
Number born dead	7		11	7		4
# Deaths  Days 1-4 (%)	12.2		21.1	19.5		7.4
# Deaths  Days 5-7 (%)	14.6		22.5	22.1		9.7
# Deaths  Days 8-14 (%)	14.9		24.7	23.2		11.8
# Deaths  Days 15-21 (%)	14.9		24.7	23.5		12.4
Mean litter size  Day 0	15.3		14.4	14.9		14.7
Day 4	14.6		12.6	13.6		13.7
Day 7	14.2		12.4	13.2		13.3
Day 14	14.2		12.1	13.1		13.0
Day 21	14.2		12.1	13.0		12.9
Parturition index	91		92	91		88
Live birth index	98		97	98		99
Viability index	89		82	82		93
Lactation index	96		92	96		95
F2 Generation
Mean Implantation Sites	17.5		17.6	17.5		16.7
Number born live	323		340	361		294
Number born dead	5		5	6		7
# Deaths   Days 1-4 (%)	8.9		12.9	6.1		4.4
# Deaths  Days 5-7 (%)	13.9		20.0	10.5		4.8
# Deaths  Days 8-14 (%)	19.5		24.1	15.2		6.8
# Deaths  Days 15-21 (%)	19.8		24.1	15.8		6.8
Mean litter size  Day 0	15.4		15.6	16.1		14.0
Day 4	14.0		13.9	15.4		13.4
Day 7	13.2		13.0	14.7		13.3
Day 14	12.4		12.3	13.9		13.0
Day 21	12.3		12.3	13.8		13.0
Parturition index	89		90	93		82
Live birth index	98		99	98		98
Viability index	91		87	91		96
Lactation index	89		84	90		98

Conclusions:

Under the conditions of this study, 10 ppm (corresponding to 1 mg/kg bw/day) was identified as a level in which only plasma cholinesterase was affected; this parameter is generally taken to indicate exposure rather than toxicity. Parental toxicity was observed at 40 and 160 ppm There were no effects on the reproductive parameters at any of the levels tested; therefore 160 ppm (corresponding to 12 and 15 mg/kg bw/day in F0 and F1, respectively) is considered a no observed adverse effect level (NOAEL) for reproductive toxicity  

Executive summary:

In a GLP compliant 2 generation reproductive toxicity study, performed according to EPA 83-4, Sprague-Dawley rats received diets containing 0, 10, 40,160 ppm of test substance. F0 animals were randomised into the 4 treatment groups, each containing 28 males and 28 females. From each treatment group, 24 male and 24 female F1 weanlings were selected for rearing to maturity and mating to produce the F2 generation. The F0 animals were treated for 10 weeks prior to mating, and then throughout the mating, gestation and lactation periods until sacrifice at the time of weaning of the F1 animals. The selected F1 animals were weaned onto the same dietary concentrations as their parents, and were maintained on these diets until termination. The F1 animals were mated at approximately 12 weeks of age. The F1 animals and their F2 litters were killed at the time of weaning of these litters.

Animals were observed for clinical signs, mortality, body weight, food consumption, mating behaviour, gestation length and degree of maternal care (including observations of pup condition and feeding). Reproduction and gestation indices were determined together with pup weight and sex ratio. There was no cull at day 4. All F0 and F1 adults received a gross post mortem examination, with reproductive organs being removed and weighed. Reproductive tissues from control and top dose animals were investigated histologically. In a later investigation (6 years later), testes from all F0 and F1 adult males were examined histologically. Sperm analyses were performed on all surviving adult males from the control and top dose groups. Gross examinations were performed on 2 pups/sex/litter at weaning and on any pups found dead or dying. No cholinesterase determinations were performed on pups.

Dietary analyses showed acceptable levels of incorporation and homogeneity, with overall achieved intakes of 0, 1, 3 or 12 mg/kg bw/d in the F0 generation and 0, 1, 4 and 15 mg/kg bw/d in F1 animals. There were no effects on mating, fertility, litter size, pup weight or pup survival in either generation, with a mean of 12 or more pups/litter in all groups at day 21. Body weight gain was reduced (~ 10%) in top dose females during gestation and lactation. Erythrocyte and brain cholinesterase activities were decreased, in a dose related manner at 40 and 160 ppm, and to a similar extent in both generations with females showing a greater sensitivity than males to inhibition of brain acetylcholinesterase. There were considered to be no adverse effects on cholinesterase activities at 10 ppm, as, with the exception of an isolated incidence in the pre-mating F1 males, the erythrocyte activity was inhibited by <20% as was the inhibition of cholinesterase activity in the brain in F0 females.

Under the conditions of this study, 10 ppm (corresponding to 1 mg/kg bw/day) was identified as a level in which only plasma cholinesterase was affected; this parameter is generally taken to indicate exposure rather than toxicity. Parental toxicity was observed at 40 and 160 ppm There were no effects on the reproductive parameters at any of the levels tested; therefore 160 ppm (corresponding to 12 and 15 mg/kg bw/day in F0 and F1, respectively) is considered a no observed adverse effect level (NOAEL) for reproductive toxicity. 

                                        

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

12 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

A gudeline study (EPA 83-4) performed in compliance with GLP.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a GLP compliant 2 generation reproductive toxicity study, performed according to EPA 83-4, Sprague-Dawley rats received diets containing 0, 10, 40,160 ppm of test substance. F0 animals were randomised into the 4 treatment groups, each containing 28 males and 28 females. From each treatment group, 24 male and 24 female F1 weanlings were selected for rearing to maturity and mating to produce the F2 generation. The F0 animals were treated for 10 weeks prior to mating, and then throughout the mating, gestation and lactation periods until sacrifice at the time of weaning of the F1 animals. The selected F1 animals were weaned onto the same dietary concentrations as their parents, and were maintained on these diets until termination. The F1 animals were mated at approximately 12 weeks of age. The F1 animals and their F2 litters were killed at the time of weaning of these litters.

Animals were observed for clinical signs, mortality, body weight, food consumption, mating behaviour, gestation length and degree of maternal care (including observations of pup condition and feeding). Reproduction and gestation indices were determined together with pup weight and sex ratio. There was no cull at day 4. All F0 and F1 adults received a gross post mortem examination, with reproductive organs being removed and weighed. Reproductive tissues from control and top dose animals were investigated histologically. In a later investigation (6 years later), testes from all F0 and F1 adult males were examined histologically. Sperm analyses were performed on all surviving adult males from the control and top dose groups. Gross examinations were performed on 2 pups/sex/litter at weaning and on any pups found dead or dying. No cholinesterase determinations were performed on pups.

Dietary analyses showed acceptable levels of incorporation and homogeneity, with overall achieved intakes of 0, 1, 3 or 12 mg/kg bw/d in the F0 generation and 0, 1, 4 and 15 mg/kg bw/d in F1 animals. There were no effects on mating, fertility, litter size, pup weight or pup survival in either generation, with a mean of 12 or more pups/litter in all groups at day 21. Body weight gain was reduced (~ 10%) in top dose females during gestation and lactation. Erythrocyte and brain cholinesterase activities were decreased, in a dose related manner at 40 and 160 ppm, and to a similar extent in both generations with females showing a greater sensitivity than males to inhibition of brain acetylcholinesterase. There were considered to be no adverse effects on cholinesterase activities at 10 ppm, as, with the exception of an isolated incidence in the pre-mating F1 males, the erythrocyte activity was inhibited by <20% as was the inhibition of cholinesterase activity in the brain in F0 females.

Under the conditions of this study, 10 ppm (corresponding to 1 mg/kg bw/day) was identified as a level in which only plasma cholinesterase was affected; this parameter is generally taken to indicate exposure rather than toxicity. Parental toxicity was observed at 40 and 160 ppm There were no effects on the reproductive parameters at any of the levels tested; therefore 160 ppm (corresponding to 12 and 15 mg/kg bw/day in F0 and F1, respectively) is considered a no observed adverse effect level (NOAEL) for reproductive toxicity.

Effects on developmental toxicity

Description of key information

- Oral: NOAEL for maternal toxicity and developmental toxicity (as a result of maternal toxicity): 15 mg/kg bw/day; rat, equivalent to OECD 414, Killick 1985

- Oral: NOAEL for maternal toxicity is 12 mg/kg bw/day, the NOAEL for developmental toxicity is 48 mg/kg bw/day15; rabbit, equivalent to OECD 414, Barton 1994

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Alpk:AP

Remarks:

Wistar-derived

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: approx. 12 weeks
- Weight at study initiation: 218-313 g
- Housing: individually housed in rat racks. The cages had solid stainless steel sides and the floor, back and front were constructed of stainless steel mesh. The internal measurements were 34.0 x 37.5 x 20.3 cm. The cages were suspended over collecting trays lined with absorbent paper.
- Diet: CT1 diet, ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 50 - 61
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was administered in corn oil. The concentration of the test substance was adjusted to give a constant dose volume of 1 mL/100 g body weight for each level dose. An appropriate amount of corn oil was added to a weighted amount of the test substance to form two 600 mL volume preparations per dose level.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A sample of each preparation was analysed prior to the start of dosing to verify the achieved concentration of the test substance in corn oil. At the end of the dosing period samples of each preparation were analysed to determine the residual concentrations of the test substance. The chemical stability of the test substance was determined by re-analysis of the dosing formulations containing nominally 0.15 and 15.0 mg/mL after an interval of 28 days. Analysis was by acetone dilution followed by gas chromatography.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant
- Proof of pregnancy: sperm in vaginal smear referred to as day 1 of pregnancy

Duration of treatment / exposure:

Days 7-16 inclusive of gestation

Frequency of treatment:

Daily

Duration of test:

15 days (gestation day 7 - 22)

Dose / conc.:

1.5 mg/kg bw/day (nominal)

Dose / conc.:

15 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

No. of animals per sex per dose:

24 females per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

All animals were dosed from Days 7-16 inclusive of gestation with 1 mL of dosing formulations per 100 g body weight using a 5 mL disposable syringe and a stainless steel 16 gauge cannula. The volume given to each animal was adjusted daily according to body weight. Control animals received the appropriate volume of corn oil. Dosing was performed in the morning of each day.

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: daily

BODY WEIGHT:
- Time schedule for examinations: Days 1 and 4 and subsequently on Days 7-16 (inclusive) and on Days 19 and 22 of gestation

FOOD CONSUMPTION:
The amount of food consumed by each animal over three day periods was measured by giving a weighed quantity of food contained in a glass jar on Days 1, 4, 7, 10, 13, 16 and 19 and calculating the amount consumed from the amount left on Days 4, 7, 10, 13, 16, 19 and 22 respectively.

POST-MORTEM EXAMINATIONS:
- One animal in the 150 mg/kg bw/day group was found dead during the study. This animal was given a macroscopic post mortem examination and pregnancy status was recorded.
- Sacrifice on gestation day # 22
- Organs examined: The intact gravid uterus (minus ovaries and trimmed free of connective tissue) was removed and weighed. The ovaries and uterus were then examined and the number of corpora lutea in each ovary as well as number and position of implantation was recorded.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: The implantations were assigned letters of the alphabet to identify their position in utero starting at the ovarian end of the left horn and ending at the ovarian end of the right horn.

Fetal examinations:

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

Statistics:

See 'Any other information on materials incl. tables'

Indices:

% Pre-implantation loss = ((No. of corpora lutea - No. of implantations)/No. of corpora lutea) x 100
% Post-implantation loss = ((No. of implantations - No. of live foetuses)/No. of implantations) x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the 150 mg/kg/day group changes in clinical condition were observed in twenty of the twenty three surviving animals. These changes included abnormal gait, changes in behaviour, signs of urinary incontinence, piloerection and body tremors and they were considered to have been induced by treatment.
There was no evidence of any adverse effects of treatment with either 1.5 or 15 mg/kg/day test substance.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

All but one animal in the 150 mg/kg/day group survived the duration of the study. This female was found dead on day 19 of gestation having shown some weight loss from day 13 and changes in clinical condition on day 16. These changes included abnormal gait, body tremors, piloerection, signs of urinary incontinence and irregular breathing. Post mortem examination revealed changes in the lungs and in the gastro-intestinal tract which may have been due to autolysis. The death of this animal was considered to be treatment-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Administration of 150 mg/kg/day test substance was associated with a statistically significant reduction in body weight gain compared with the control group during the dosing and the post dosing period. The effect was most marked between days 13 and 16 of the dosing period and days 16 and 19 of the post dosing period. Between days 19 and 22, weight gain was comparable with the control group.
There were no adverse effects of treatment on maternal weight gain with either 1.5 or 15 mg/kg/day test substance

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Administration of 150 mg/kg/day test substance was associated with a statistically significant reduction in food consumption compared with the control group for the dosing period and the post dosing period. The effect attained statistical significance from day 13 onwards.
There were no adverse effects of treatment on maternal food consumption with either 1.5 or 15 mg/kg/day test substance.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

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):

Only two animals in the 1.5 mg/kg/day group surviving to termination showed any macroscopic changes post mortem. One female had bilateral renal pelvic dilatation and another female had dark red fluid surrounding the foetuses in utero. These changes were of the type commonly seen in the Alpk:AP strain of rat and were considered not to be treatment-related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

In the 1.5 mg/kg/day group, pre-implantation loss was statistically significantly increased compared with the control group. Consequently, the mean number of implantations and of live foetuses and hence, mean gravid uterus and litter weight, were all statistically significantly reduced. These values were nevertheless within the range expected for control rats of this strain and in the absence of a dosage-related trend, these observations were considered to be of no toxicological significance.

Total litter losses by resorption:

not examined

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

mortality

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:

effects observed, non-treatment-related

Description (incidence and severity):

In the 150 mg/kg/day group, the proportion of foetuses which were male was statistically significantly increased in comparison with the control when analysed on a foetus basis but not when analysed on a litter basis. The values for all groups including the control group were however, within the historical control range for this strain of rat and therefore, no adverse effects of treatment with the test substance were considered likely.

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):

The incidence of foetuses with major defects was 1, 0, 0 and 2 in the control, 1.5, 15 and 150 mg/kg bw/day groups, respectively. The defects were not similar in type and there was no evidence to suggest that their occurence was due to treatment with the test substance.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The incidence of foetuses with fully ossified 4th lumbar transverse processes was statistically significantly increased in the 1.5 and 150 mg/kg/day groups in comparison with the control group. In the absence of any dosage-relationship, this finding was considered to be unrelated to treatment. The mean manus and pes scores for the treated groups were not statistically significantly different from the control group. The pes scores for the
150 mg/kg/day group did, however, reflect a very slight reduction in ossification compared with the control group.

Visceral malformations:

no effects observed

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

150 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

Relevant for humans:

yes

Table 1. Clinical signs of toxicity seen in dams

Dose (mg/kg bw/d)	0	1.5	15	150
Number examined	24	24	24	24
Maternal b wt gain (g)	152.5	149.3	157.9	109.5**
Maternal food consumption (g/day)	515.5	513.7	524.5	433.7**
	Number showing effect
Increased activity	0	0	0	1
Increased response to touch	0	0	0	1
Splayed gait	0	0	0	4
Tip toe gait	0	0	0	4
Reduced stability	0	0	0	2
Tremors	0	0	0	7
Hunched	0	0	0	1
Subdued	0	0	0	6
Decreased abdominal tone	0	0	0	1
Piloerection	1	0	3	14
Irregular breathing	0	0	0	2
Signs of salivation	0	0	0	1
Stains around nose	0	0	0	4
Signs of urinary incontinence	0	0	0	13

 

Table 2. Foetal ‘Pes’ scores

Dose (mg/kg bw/d)	0	1.5	15	150
Mean Pes score	2.93	2.78	2.92	3.07

Conclusions:

There was no evidence of teratogenicity in this study with any of the dose levels of the test substance used. Administration of 150 mg/kg bw/day was associated with maternal toxicity and with minimal foetotoxicity.
There were no adverse effects of treatment with either 1.5 or 15 mg/kg bw/day test substance, therefore, the NOAEL for maternal toxicity and teratogenicity is 15 mg/kg bw/day.

Executive summary:

In a GLP compliant study, comparable to OECD TG 414, groups of 24 pregnant female rats were dosed by gavage with 0, 1.5, 15 or 150 mg/kg bw/day test substance in corn oil from days 7-16 (inclusive) of gestation which thus included the period of major organogenesis. The day of confirmation of mating was designated Day 1 of gestation. On Day 22 of gestation the females were killed and their uteri examined for live foetuses and intra-uterine deaths.

The foetuses were weighed, examined for external and visceral abnormalities, sexed, eviscerated and stained for skeletal examination. Administration of 150 mg/kg bw/day test substance was associated with maternal toxicity manifest as treatment-related changes in clinical condition, reduced bodyweight gain and food consumption. There was no evidence of maternal toxicity associated with either 1.5 or 15 mg/kg bw/day administration of the test substance. There were no effects of the test substance on the number, growth and survival of the young in utero with any of the dose levels used. There was no evidence of teratogenicity or foetotoxicity associated with the administration of 1.5, 15 or 150 mg/kg bw/day test substance as assessed by the type and incidence of major defects and the incidence of minor defects and variants. There was however, some minimal evidence of foetotoxicity with 150 mg/kg bw/day dose manifest as marginal reduction in the ossification of the pes. In conclusion, administration of 150 mg/kg bw/day test substance was associated with maternal toxicity and with minimal evidence of foetotoxicity (as a result of maternal toxicity). There were no adverse effects of treatment with either 1.5 or 15 mg/kg bw/day test substance, therefore, the NOAEL for maternal toxicity and teratogenicity is 15 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

15 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

A guideline study (comparable to OECD 414) performed in compliance with GLP

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Developmental toxicity study in rats, Killick 1985

In a GLP compliant study, comparable to OECD TG 414, groups of 24 pregnant female rats were dosed by gavage with 0, 1.5, 15 or 150 mg/kg bw/day test substance in corn oil from days 7-16 (inclusive) of gestation which thus included the period of major organogenesis. The day of confirmation of mating was designated Day 1 of gestation. On Day 22 of gestation the females were killed and their uteri examined for live foetuses and intra-uterine deaths. The foetuses were weighed, examined for external and visceral abnormalities, sexed, eviscerated and stained for skeletal examination. Administration of 150 mg/kg bw/day test substance was associated with maternal toxicity manifest as treatment-related changes in clinical condition, reduced body weight gain and food consumption. There was no evidence of maternal toxicity associated with either 1.5 or 15 mg/kg bw/day administration of the test substance. There were no effects of the test substance on the number, growth and survival of the young in utero with any of the dose levels used. There was no evidence of teratogenicity or foetotoxicity associated with the administration of 1.5, 15 or 150 mg/kg bw/day test substance as assessed by the type and incidence of major defects and the incidence of minor defects and variants. There was however, some minimal evidence of foetotoxicity with 150 mg/kg bw/day dose manifest as marginal reduction in the ossification of the pes. In conclusion, administration of 150 mg/kg bw/day test substance was associated with maternal toxicity and with minimal evidence of foetotoxicity (as a result of maternal toxicity). There were no adverse effects of treatment with either 1.5 or 15 mg/kg bw/day test substance, therefore, the NOAEL for maternal toxicity and teratogenicity is 15 mg/kg bw/day.

Developmental toxicity study in rabbits, Barton 1994

In a GLP compliant study comparable to OECD 414, the developmental toxicity of the test substance was investigated in rabbits. Groups of 16 mated, female New-Zeeland White rabbits received the test substance by gavage in corn oil at 0, 12, 24 or 48 mg/kg bw/day on days 6 - 18 of gestation. Animals were sacrificed on day 29 of gestation and the uterine contents investigated for resorptions, viable foetuses, foetal weight, plus external, visceral and skeletal abnormalities. Cholinesterase determination were performed on samples of blood taken on the mornings of days 5, 19 and 29 of gestation and on brain samples taken at necropsy of the same 6 animals per group. The only pre-terminal death was a mid-dose animal sacrificed following abortion. Maternal signs, body weight, food consumption and pregnancy outcome and basic foetal parameters were unaltered by treatment. The only foetal finding was a slight increase in the number of foetuses showing pelvic shift at the top dose (0.9% in controls vs 3.6% for cephlad shift and 8% vs 10.7% for caudal shift). This finding showed no dose response and there was no statistical difference between the findings in control and high dose animals (Fisher’s exact test). When the number of litters is considered, there is no difference between the control and high dose groups for caudal shift (three litters each) while cephalad shift is seen in one litter in the control and two litters at the top dose. As with the foetal findings there is no dose response and the incidence in the top dose group is not statistically different from the control groups. Given the high incidence of pelvic shift and comparable litter numbers in controls this is most likely to be a chance finding unrelated to treatment. Furthermore, this finding is considered to be a variation rather than a malformation and was only seen at a maternally toxic dose. The finding of 4 microphthalmias in the same mid-dose litter is not considered to be treatment related as microphthalmias were not seen at the top dose level nor in the sighting study at doses up to 32 mg/kg bw/day. Erythrocyte cholinesterase activity was inhibited in all test groups on day 19 of gestation, persisting until day 29 at 48 mg/kg bw/day. Brain cholinesterase activity was inhibited at 48 mg/kg bw/day on day 29 of gestation (11 days after the last dose).

The inhibition of RBC cholinesterase in the 12 mg/kg bw/day group on day 19 (21%) does not achieve statistical significance and is on the borderline for the accepted value considered as a marker of adversity (20%). In the absence of other effects at this dose, 12 mg/kg bw/day is considered to be the NOAEL for maternal toxicity. As there was no developmental toxicity up to the top dose, the NOAEL for developmental toxicity is therefore 48 mg/kg bw/day.

Justification for classification or non-classification

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