imidacloprid (ISO); 1-(6-chloropyridin-3-ylmethyl)-N-nitroimidazolidin-2-ylidenamine

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

2-generation (rat, OECD 416):

NOAEL (systemic, P0/F1): 250 ppm (correspondonding to ca. 20 mg/kg bw/day during premating period)

LOAEL (systemic, P0/F1): 700 ppm (correspondonding to ca. 50 mg/kg bw/day during premating period)

NOAEL (fertility, P0/F1): ≥700 ppm (correspondonding to ca. 40 mg/kg bw/day during premating and pregnancy)

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 Nov 1987 - 17 Feb 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

adopted 2001

Deviations:

yes

Remarks:

Please refer to "Principles of method if other then guideline"

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

adopted 1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

Version / remarks:

adopted 1984

Deviations:

no

Principles of method if other than guideline:

The study differs from the actual version of the OECD test guideline in the following points: oestrus cycle as well as sperm parameters were not evaluated, pairs without progeny were not evaluated to determine cause of infertility, age of vaginal opening and preputial separation for F1 weanings selected for mating was not recorded, data on physical landmarks in pups and other postnatal developmental data were not recorded, uteri of primiparous females were not examined for presence of implantation sites, brains of parental animals and offspring were not weighed, spleen and thymus of offspring were not weighed.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Common strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: KFM, Kleintierfarm Madoerin AG, Fuellinsdorf, Switzerland
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 5 weeks; (F1) 3 weeks
- Weight at study initiation: (P) Males: 123-169 g; Females: 81-137 g; (F1) Males: 176-298 g; Females: 132-213 g
- Fasting period before study: not applicable
- Housing: individually in Makrolon type-3 cages with wire mesh tops and standard granulated, softwood bedding (Lignocel, Schill AG, Muttenz, Switzerland), cohoused during pairing
- Diet: pelleted standard Kliba 343, rat/mouse maintenance diet (Klingentalmuehle AG, Kaiseraugst, Switzerland), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 10 days

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: 23 Oct 1987 To: 17 Feb 1989

Route of administration:

oral: feed

Mass median aerodynamic diameter (MMAD):

other: not applicable

Remarks on MMAD:

not applicable

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: at least every 2 weeks
- Mixing appropriate amounts with granulated food: The test article was mixed to granulated food in a Buehler Mixer, type DDMA-0.5 and pelleted in a Buehler pelleting machine type DFPL (Buehler Ltd., Uzwil, Switzerland). Water was added to each feed preparation at an approximately 1:10 volume/weight ratio to ensure pelleting, after which the pellets were dried with warm air for 48-96 hours before storage.
- Storage temperature of food: room temperatue in the dark

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: maximum 22 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 post coitum
- After 22 days of unsuccessful pairing replacement of first male by another male.
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of the test article concentration, its homogeneity and stability in the food pellets was determined before starting the study with the dose finding study using HPLC.
The content and homogeneity of the test article in the food pellets was determined at start of the prepairing periods, mating and at the end of gestation/start of lactation periods. The test substance was stable for 3 weeks in the diet. The overall mean concentrations were 98.1 %, 97.1 % and 97.7 % of the nominal concentration for the 100 ppm, 250 ppm and 700 ppm dose group, respectively. The homogeneity varied in the range of -10 % to +9 % of the analyzed feed batches.

Duration of treatment / exposure:

P generation
during a 84-day prepairing period and also during the pairing-, gestation- and lactation periods for breeding the F1A and F1B litters.

F1 animals
starting at 7 or 8 weeks of age, during their growth into adulthood (105-days prepairing period) and also during the pairing-, gestation- and lactation periods for breeding the F2A and F2B litters

Frequency of treatment:

daily, 7 days/week

Details on study schedule:

- F1 parental animals not mated until 18 weeks after selected from the F1 litters (after 105 days of feeding with test substance)
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 17 weeks (P generation for F1A litters), 28 weeks (P generaion for F1B litters), 22 weeks (F1 parents for F2A litters), ca 28-31 weeks (F1 parents for F2B litters)

Dose / conc.:

100 ppm (nominal)

Remarks:

corresponding to actual dose ingested:
5-13 mg/kg bw/day for P generation males (P0),
6-13 mg/kg bw/day for P generation females (P0) during prepairing and gestation,
10-18 mg/kg bw/day for P generation females (P0) during lactation,
5-9 mg/kg bw/day for F1 parental males (P1),
6-9 mg/kg bw/day for F1 parental females (P1) during prepairing and gestation,
11-18 mg/kg bw/day for F1 parental females (P1) during lactation

Dose / conc.:

250 ppm (nominal)

Remarks:

corresponding to actual dose ingested:
12-32 mg/kg bw/day for P generation males (P0),
16-32 mg/kg bw/day for P generation females (P0) during prepairing and gestation,
25-46 mg/kg bw/day for P generation females (P0) during lactation,
12-23 mg/kg bw/day for F1 parental males (P1),
16-24 mg/kg bw/day for F1 parental females (P1) during prepairing and gestation,
26-42 mg/kg bw/day for F1 parental females (P1) during lactation

Dose / conc.:

700 ppm (nominal)

Remarks:

corresponding to actual dose ingested:
35-89 mg/kg bw/day for P generation males (P0),
44-96 mg/kg bw/day for P generation females (P0) during prepairing and gestation,
73-121 mg/kg bw/day for P generation females (P0) during lactation,
36-68 mg/kg bw/day for F1 parental males (P1),
41-68 mg/kg bw/day for F1 parental females (P1) during prepairing and gestation,
78-120 mg/kg bw/day for F1 parental females (P1) during lactation

No. of animals per sex per dose:

30 (P generation (P0))
26 (F1 parental animals (P1))

Control animals:

yes

Details on study design:

- Dose selection rationale: Doses were selected based on a previous range finding experiment (M-027080-01-2). There, 10 male and 10 female rats were fed with 0, 20, 100 or 500 ppm of the test substance in pelleted diet. Administration was continued for a three-week prepairing period and throughout the pairing, gestation and lactation periods. Pups of the F1 generation were reared for a further week on the respective test diet after weaning (from post partum Day 21). It was shown that at 500 ppm, P females had a reduced body weight gain during the preparing period and consequently decreased mean body weights during the following gestation and lactation periods, they also consumed less food than control animals during the lactation period. In the F1 generation, decreased mean body weight on day 0 post partum and throughout the lactation and rearing period were observed in the 500 ppm group as well as reduced food consumption during the rearing period (days 21 - 28 post partum). Therefore, the NOEL of the test substance was set to 100 ppm and the dose rationale for the main study was set to 100, 250 and 700 ppm.
- Fasting period before blood sampling for clinical biochemistry: no

Positive control:

No

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
Animals were examined for clinical signs of reaction related to the treatment, affecting behaviour and general appearance.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly (with exception of pairing periods), after mating on days 0, 7, 14, and 21 and on days 0, 4, 7, 14 and 21 post partum

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
- Time schedule: weekly (with exception of pairing periods). During the lactation periods, food consumption was recorded only till day 14 post partum


OTHER:
Blood samples for hematology and clinical biochemistry were collected from 10 randomly selected animals per group and sex of the F1 generation prior to necropsy. Parameters checked were erythrocyte count (RBC), hemoglobin (HB), hematocrit (HCT), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count, reticulocyte count (retic.), nucleated erythrocytes normoblasts (NEN), total leucocyte count (WBC), differential leukocyte count (diif. WBC count, analyzed for band neutrophils, segmented neutrophils, eosinophils, basophils, lymphocytes, monocytes, plasma cells, blast cells), red cell morphology, thromboplastin time (PT), partial thromboplastin time (PTT), glucose, urea, creatinine, bilirubin total (Bili-T), total lipids, total cholesterol, triglycerides, phospholipids, aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), lactate dehydrogenase (LDH), creatine kinase (CK), alkaline phosphatase (ALP), gamma-gutamyl-transferase (GGT), calcium, phosphorus, sodium, potassium, chloride, total protein, protein electrophoresis

Samples of liver were taken from all animals mentioned above at necropsy for the assay of triglycerides, cytochrome P-450 and N-and 0-demethylase activity.

Oestrous cyclicity (parental animals):

Mating data were considered for detection of anomalies affecting the estrus cycle of the females.

Sperm parameters (parental animals):

Parameters examined in male parental generations:
- males that failed to induce pregnancy: testis weight, epididymides weight, histopathological examination of testes, epididymides, prostate and seminal vesicles
- all animals of control and high dose group: histopathological examination of testes, epididymides, prostate and seminal vesicles

F1 parent animals:
- males that failed to induce pregnancy: testis weight, epididymides weight, histopathological examination of testes, epididymides, prostate and seminal vesicles

Litter observations:

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

PARAMETERS EXAMINED
The following parameters were examined in F1and F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, physical or behavioural abnormalities, weight gain (F1A and F1B pups were weighed individually on days 0, 4, 7, 14 and 21 of lactation. F2A and F2B pups were weighed individually on days 0 (if possible) and/or 1, 4, 7, 14 and 21 of lactation)

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

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were sacrificed, exact time point is not reported
- Maternal animals: All surviving animals where sacrificed after the weaning of the F1B litter

GROSS NECROPSY
- Gross necropsy consisted of all gross lesions, liver, ovaries, pituitary gland, prostate, seminal vesicles with coagulating gland, thyroid gland, testes with epididymides, uterus and cervix, vagina

HISTOPATHOLOGY / ORGAN WEIGHTS
- tissues prepared for microscopic examination and weighed, respectively: liver, ovaries, pituitary gland, prostate, seminal vesicles with coagulating gland, thyroid gland, testes with epididymides, uterus and cervix, vagina

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age.
- These animals were subjected to postmortem examinations as follows: all pups were examined macroscopically, from one male and one female per litter specified organs were weighed and tissues preserved for histopathological examination

GROSS NECROPSY
- Gross necropsy consisted of all gross lesions, liver, ovaries, pituitary gland, prostate, seminal vesicles with coagulating gland, thyroid gland, testes with epididymides, uterus and cervix, vagina

HISTOPATHOLOGY / ORGAN WEIGHTS
- tissues prepared for microscopic examination and weighed, respectively: liver, ovaries, pituitary gland, prostate, seminal vesicles with coagulating gland, thyroid gland, testes with epididymides, uterus and cervix, vagina

Statistics:

If the variable could be assumed to follow a normal distribution: Univariate one-way analysis of variance was used.
If significance was noted, Dunnett many-one t-test was used for inter-group comparison.
For reproduction parameters, An one-way univariate analysis of variance based on Wilcoxon ranks together with the Kruskall-Wallis test was applied.
For the spontaneous mortality of pups data, Fisher's Exact test for 2x2 tables was used.

Reproductive indices:

mean precoital time, fertility index, conception rate and gestation index

Offspring viability indices:

not reported

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

incidental findings were observed, mainly hair loss and wounds but no dose-relationship was noticed and findings were occasional. Therefore, they were considered incidental.

Dermal irritation (if dermal study):

no effects observed

Description (incidence and severity):

not applicable

Mortality:

mortality observed, non-treatment-related

Description (incidence):

- Control: 1 female was found dead on Day 38 of the preparing period and 1 female was killed for humane reasons on day 21 post coitum breeding for F1B litters because of poor condition.
- 100 ppm: 1 female was found dead on Day 36 of the preparing period.

Since no dose-response was observable, these deaths were considered incidental.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males
- 700 ppm: reduced mean body weight in the preparing period compared to controls (from Day 8 on, from -5.2 to -9.1%), the body weight gain was similar to controls during post-paring period but the significantly lower mean body weights remained.

Females
Prepairing period for breeding F1A litters
- 100 ppm: statistically significant increased body weight to controls on Day 1 (+4.5%), considered incidental
- 250 ppm: statistically significant increased body weight to controls on Day 1 (+4.5%), considered incidental
- 700 ppm: reduced body weight to controls throughout the prepairing period with statistical significance on days 29, 36, 43, 57, 71 and 78 (from -5.5 to -7.0%)

Gestation period for breeding F1A litters
- 700 ppm: body weight remained lower than controls (statistically significant up to Day 14, from -6.9 to -7.0%) but body weight gain was similar to control animals, therefore this was not considered related to treatment.

Lactation period for breeding F1A litters
- 700 ppm: statistically significant reduced body weight compared to controls until Day 7 (from -6.9 to -7.2%)

Gestation period for breeding F1B litters
- 700 ppm: statistically significant reduced body weight compared to controls throughout gestation period (from -4.9 to -5.8%) but body weight gain similar to controls (42.5 vs 42.9% in controls)

Lactation period for breeding F1B litters
- 700 ppm: statistically significant lower mean body weight at Day 0 (-5.5%), increased body weight gain compared to controls

Summarized data can be found in Table 1 (Attachment 1) in the attached background material.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males:
- 700 ppm: reduced food consumption during preparing period for F1A litters (statistically significant compared to controls for days days 8-15 (-5.7%), 22-57 (from -6.3 to -9.5%) and 64 - 78 (from -5.7 to -6.0%)

Females
Prepairing period for breeding F1A litters
- 700 ppm: reduced compared to controls from Day 8 to Day 84 (statistically significant different to controls from Days 22-43 and 64-71 with reduction from -7.7 to -8.8 and -7.1, respectively)

Gestation period for breeding F1A litters
- 700 ppm: statistically significant reduced food consumption throughout the gestation period (between -7.5 and -8.0%) compared to controls

Lactation period for breeding F1A litters
- 700 ppm: reduced food consumption throughout the lactation period (statistically significant from Day 4 to Day 14 with reduction from -10.4 and -11.5%) compared to controls

Gestation period for breeding F1B litters
- 700 ppm: statistically significant reduced food consumption throughout the gestation period (between -7.7 and -12.1%) compared to controls

Lactation period for breeding F1B litters
- 700 ppm: reduced food consumption throughout the lactation period (statistically significant from Day 7 to Day 14 with reduction from -9.3%)

Summarized data can be found in Table 2 (Attachment 2) within the attached background material.

Food efficiency:

not examined

Description (incidence and severity):

not applicable

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

not examined

Description (incidence and severity):

not applicable

Ophthalmological findings:

not examined

Description (incidence and severity):

not applicable

Haematological findings:

not examined

Description (incidence and severity):

not applicable

Clinical biochemistry findings:

not examined

Description (incidence and severity):

not applicable

Endocrine findings:

not examined

Description (incidence and severity):

not applicable

Urinalysis findings:

not examined

Description (incidence and severity):

not applicable

Behaviour (functional findings):

not examined

Description (incidence and severity):

not applicable

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No test article related changes were noted at the histological examination of the coagulating glands, epididymides, liver, ovaries, pituitary glands, prostate gland, seminal vesicles, testes, thyroid glands, uterus and vagina in control and high dose animals.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

not applicable

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

not applicable

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

not applicable

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating performance, fertility, duration of gestation, litter size at first litter check, postnatal loss from Day 0 to Day 4 post partum and breeding loss from Day 4 to Day 21 post partum were similar throughout all groups including the control.

Non treatment related findings were
- 700 ppm: statistical significant more pubs per dam in F1B litter but within the historical control range, one female lost its entire F1B litter between days 6 and 12 of the lactation period (considered incidental)

There was no evidence of an adverse effect of treatment with on any of the reproduction indices: mean precoital time, fertility index, conception rate and gestation index.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

250 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at this concentration

Remarks on result:

other: corresponding to 20 mg/kg bw/day

Key result

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

700 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: corresponding to 50 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

>= 700 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed up to the highest concentration tested

Remarks on result:

other: corresponding to 40 mg/kg bw/day

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs of reaction related to treatment.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality:

mortality observed, non-treatment-related

Description (incidence):

- 100 ppm: one male died after blood sampling at the end of the study (no abnormal findings were noted at necropsy).
- 250 ppm: one male was killed for human reasons on day 14 of the post-pairing period for breeding F2B litters because of poor condition.

These deaths were considered incidental.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Males
- 700 ppm: since body weight was reduced in pups of this group at weaning, mean body weight was reduced between Days 1 and 22 of the prepairing period but increased thereafter. Body weight gain was slightly higher increased compared to controls during the prepairing period or similar during the post-pairing period compared to controls.

Therefore, the findings were considered incidental.

Females
Prepairing period for breeding F2A litters
- 700 ppm: body weight was reduced throughout the prepairing period compared to controls (from -7.5 to -9.3%)

Gestation period for breeding F2A litters
- 700 ppm: reduced mean body weight was continued and statistically significant throughout the gestation period compared to controls (from -10.0 to -10.2%) but body weight change was similar to control group.

Lactation period for breeding F2A litters
- 700 ppm: reduced mean body weight was continued and statistically significant throughout the gestation period compared to controls (from -6.8 to -11.2%) but body weight change was similar to control group.

Gestation period for breeding F2B litters
- 700 ppm: reduced mean body weight was continued and statistically significant throughout the gestation period compared to controls (from -7.9 to -9.6%) but body weight change was similar to control group.

Lactation period for breeding F2B litters
- 700 ppm: reduced mean body weight was continued and statistically significant throughout the gestation period (from -5.7 to -10.9%) but body weight change was similar to control group.

The reduced body weight of females was considered a secondary effect of the lower body weights noted from the F1B pups compared to controls at weaning and therefore not directly related to treatment with the test article. Summarized Data can be found in Table 3 (Attachment 3) within the attached background material.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Males
-100 ppm: food consumption was higher than in controls, statistical significance was found for the period between Day 1-8 of the preparing period (+6.7%) but considered incidental
- 700 ppm: slightly increased relative food consumption compared to control group during some weeks of the prepairing period but considered incidental.

Females
Prepairing period for breeding F2A litters
- 700 ppm: reduced statistically significant compared to controls from Days 15-29 (from -7.7 to -11.8%) and 57-105 (from -5.4 to -16.0%)

Gestation period for breeding F2A litters
- 700 ppm: statistically significant reduced food consumption compared to controls throughout the gestation period (between -11.0 and -13.8%)

Lactation period for breeding F2A litters
- 100 ppm: slightly increased food consumption (between +4.3 and +7.0%) but not statistically significant compared to controls
- 700 ppm: slightly reduced food consumption from Day 1 to 14 (from -2.6 to -6.6%) but without statistical significance compared to controls

Gestation period for breeding F2B litters
- 250 ppm: reduced food consumption compared to controls from Day 0-7 (-7.8%) and Day 7-14 (-6.8%)
- 700 ppm: statistically significant reduced food consumption compared to controls throughout the gestation period (between -10.7 and -13.7%)

Lactation period for breeding F2B litters
- 250 ppm: reduced food consumption but without statistical significance compared to controls (from -4.0 to -8.4%)
- 700 ppm: reduced food consumption but statistical significance compared to controls only from Day 7-14 (-9.5%)

The findings for 250 ppm were considered incidental because they were probably mostly caused by the slightly reduced mean number of pups noted in this group.

Summarized data can be found in Table 4 (Attachment 4) within the attached background material.

Food efficiency:

not examined

Description (incidence and severity):

not applicable

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

not examined

Description (incidence and severity):

not applicable

Ophthalmological findings:

not examined

Description (incidence and severity):

not applicable

Haematological findings:

no effects observed

Description (incidence and severity):

All statistical differences in the results of the haematology parameters were considered to be incidental and of normal variation for rats of this strain and age.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- 250 ppm: slight but statistical significant increase in O-demethylase activity compared to controls (23% in females, seen as adaptive proicess as described below)
- 700 ppm: slight but statistical significant increase in hepatic Cytochrome P-450 content (26%, males) and N-demethylase activity (18%, males), slight increase in O-demethylase activity (36% males/37%, females), all compared to controls

These findings may indicate a slight stimulation of mixed-function oxidase activities. However, other markers for liver toxicity were not observed (namely liver-derived plasma enzymes, plasma bilirubin, liver triglycerides or any effect on liver morphology).

Summarized data can be found in Table 5 in Attachment 5.

Endocrine findings:

not examined

Description (incidence and severity):

not applicable

Urinalysis findings:

not examined

Description (incidence and severity):

not applicable

Behaviour (functional findings):

not examined

Description (incidence and severity):

not applicable

Immunological findings:

not examined

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- 700 ppm: statistically reduced absolute ovary weights (-13.5%) compared to controls. However, the ovaries did not show any other abnormal macroscopical or histopathological changes and the relative ovary weights were similar to those of the control females. Thus, this finding was considered to be a secondary effect of the reduced mean body weight and not considered directly treatment related.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item related macroscopic findings were observed in any of the treatment groups.

Neuropathological findings:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No test article related changes were noted at the histological examination of the coagulating glands, epididymides, liver, ovaries, pituitary glands, prostate gland, seminal vesicles, testes, thyroid glands, uterus and vagina in control and high dose animals.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

not applicable

Other effects:

not examined

Description (incidence and severity):

not applicable

Reproductive function: oestrous cycle:

not examined

Description (incidence and severity):

not applicable

Reproductive function: sperm measures:

not examined

Description (incidence and severity):

not applicable

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Mating performance, fertility, duration of gestation, litter size at first litter check, postnatal loss from Day 0 to Day 4 post partum and breeding loss from Day 4 to Day 21 post partum were similar throughout all groups including the control.

Non treatment related findings were
- 250 ppm: breeding loss in F2A litters from day 4 to day 21 post partum was statistically increased (4.7% vs 0.7% in controls). This was considered incidental and within the normal range for this strain of rat. In F2B litters, the litter size was slightly reduced (9.2 vs 10.8 in controls) but no dose relationship was found.

There was no evidence of an adverse effect of treatment on any of the following reproduction indices: mean precoital time, fertility index, conception rate and gestation index.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

250 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed up to this concentration

Remarks on result:

other: corresponding to 20 mg/kg bw/day

Key result

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

700 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: corresponding to 50 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

>= 700 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed up to the highest concentration tested

Remarks on result:

other: corresponding to 40 mg/kg bw/day

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

not applicable

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

- Control: one F1A pub was found dead after parturition and showed cheiloschisis.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 700 ppm: in F1A litters, the body weight gain was reduced during the lactation period compared to controls, this was statistically significant from Day 4, resulting in reduced mean body body weight at Day 21(-13.4%). In F2A litters, the body weight gain was reduced during the lactation period, this was statistically significant compared to controls from Day 7, resulting in reduced mean body weight at Day 21(-9.6%) compared to controls.

Summarized data can be found in Attachmend 6.

Food consumption and compound intake (if feeding study):

not examined

Description (incidence and severity):

not applicable

Food efficiency:

not examined

Description (incidence and severity):

not applicable

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

not examined

Description (incidence and severity):

not applicable

Ophthalmological findings:

not examined

Description (incidence and severity):

not applicable

Haematological findings:

not examined

Description (incidence and severity):

not applicable because only evaluated in F1 parental animals (=P1)

Clinical biochemistry findings:

not examined

Description (incidence and severity):

not applicable because only evaluated in F1 parental animals (=P1)

Urinalysis findings:

not examined

Description (incidence and severity):

not applicable

Sexual maturation:

not examined

Description (incidence and severity):

not applicable

Anogenital distance (AGD):

not examined

Description (incidence and severity):

not applicable

Nipple retention in male pups:

not examined

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- 700 ppm: absolute weights of the liver was reduced in F1A (-17.3% males, -14.6% females) and F1B (12.9% males, -8.6% females) pups compared to controls, absolute weights of testes or ovaries were reduced in F1A pups (-12% males, -25% females), testes/body weight ratio was increased in male F1B pups (+4.5%) compared to controls.

These findings were considered incidental because they were not seen in the other generation and showed no correlation with the reduced mean body weight.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item related macroscopic findings were observed in any of the treatment groups.

Histopathological findings:

not examined

Description (incidence and severity):

not applicable

Other effects:

not examined

Description (incidence and severity):

not applicable

Behaviour (functional findings):

not examined

Description (incidence and severity):

not applicable

Developmental immunotoxicity:

not examined

Description (incidence and severity):

not applicable

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

250 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed up to the highest concentration tested

Remarks on result:

other: corresponding to ca. 40 mg/kg bw/day (during lactation)

Key result

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

700 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: corresponding to 100 mg/kg bw/day (during lactation)

Key result

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

- 250 ppm: one female pup showed a stumped tail at external examination at first litter check.

Dermal irritation (if dermal study):

no effects observed

Description (incidence and severity):

not applicable

Mortality / viability:

no mortality observed

Description (incidence and severity):

not applicable

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 700 ppm: in F2A litters, the body weight gain was reduced compared to controls during the lactation period, this was statistically significant from Day 7, resulting in reduced mean body body weight at Day 21(-9%). In F2B litters, the body weight gain was reduced throughout the lactation period, this was statistically significant compared to controls from Day 7, resulting in reduced mean body body weight at Day 21(-13.4%).

Summarized data can be found in Attachment 7.

Food consumption and compound intake (if feeding study):

not examined

Description (incidence and severity):

not applicable

Food efficiency:

not examined

Description (incidence and severity):

not applicable

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

not examined

Description (incidence and severity):

not applicable

Ophthalmological findings:

not examined

Description (incidence and severity):

not applicable

Haematological findings:

not examined

Description (incidence and severity):

not applicable

Clinical biochemistry findings:

not examined

Description (incidence and severity):

not applicable

Urinalysis findings:

not examined

Description (incidence and severity):

not applicable

Sexual maturation:

not examined

Description (incidence and severity):

not applicable

Anogenital distance (AGD):

not examined

Description (incidence and severity):

not applicable

Nipple retention in male pups:

not examined

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- 250 ppm: increase of the absolute and relative weight of the ovaries in F2A litter (23.5% and 23.0%, respectively) as compared to controls.
- 700 ppm: decreased liver weight in F2B males (-10.3%) but similar liver/body weight ratio as in control groups


The increased ovary weight was considered incidental since no dose-relationship was observed and the finding was not seen in the F2B litter. The decreased liver weight is considered a result of the reduced body weight and therefore not directly treatment related.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test article realted findings were observed. In the pups that were killed 4 days post partum, occasional findings included reduced kidney weight, increased kidney size, agnesia of the left testis and worms in the intestine. These findings were spred through all treatment and control groups, no dose-relationship was found and they were therefore considered incidental.

Histopathological findings:

no effects observed

Description (incidence and severity):

No test article related changes were noted at the histological examination of the coagulating glands, epididymides, liver, ovaries, pituitary glands, prostate gland, seminal vesicles, testes, thyroid glands, uterus and vagina in control and high dose animals.

Other effects:

not examined

Description (incidence and severity):

not applicable

Behaviour (functional findings):

not examined

Description (incidence and severity):

not applicable

Developmental immunotoxicity:

not examined

Description (incidence and severity):

not applicable

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

250 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at this concentration

Remarks on result:

other: corresponding to 40 mg/kg bw/day

Key result

Dose descriptor:

LOAEL

Generation:

F2

Effect level:

700 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: corresponding to 100 mg/kg bw/day

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

To assess reproductive toxicity, a 2-generation study was conducted according to OECD TG 416 with male and female rats. The test item was offered to the animals via feed, at doses of 100, 250 and 700 ppm. No treatment-related mortality occurred. As compared to the untreated controls, reduced food consumption was observed for P0 and P1 animals that received 700 ppm of the test substance during the prepairing period. For females, reduced food consumption was also observed at 700 ppm during all gestation and lactation periods (P0 and P1 animals). Consequently, animals of the 700 ppm group had reduced body weight. The body weight gain was especially reduced during the prepairing period of P0 animals (male and female), after that, it was similar to controls but body weight remained low because of the established weight difference. In all offspring generations of the 700 ppm dose group (F1A, F1B, F2A and F2B), reduced body weight gain was observed. In the P1 generation, increased O-demethylase activity in the liver was observed for females receiving 250 ppm test substance and both sexes of the 700 ppm group. Also, hepatic N-demethylase activity and hepatic cytochrome P-450 content was increased in males at 700 ppm. This indicates increased xenobiotic metabolism but no further evidence of liver toxicity was found. External examination of pups revealed no obvious abnormalities indicative of teratogenicity in any group of either generation. Therefore, the NOAEL for parental toxicity can be set at 250 ppm for male and female rats under the conditions of the study. The NAOEL for fertility was >= 700 ppm, since no alteration of reproduction parameters was observed and the NOAEL for developmental toxicity was set at 250 ppm.

The study was conducted according to GLP and in accordance with the OECD Guideline 416 in the version of 1981.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

40 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

The reproduction toxicity was assessed in a 2-generation study conducted with male and female rats according to OECD TG 416 (1983), under GLP conditions. The study is considered of reliable quality and validity, fulfills the criteria of a key study and thus, is suitable for assessment of the present endpoint.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

To assess the effect on reproduction of the test item, a 2-generation key study conducted with rat as well as two developmental toxicity key studies, one conducted with the rat as rodent and one conducted with the rabbit as non-rodent species, are available. For each study, a range-finding preliminary experiment was conducted.

 

In the 2-generation reproductivity study, rats were fed with the test substance over two generations; all parental animals (P0 and P1) received the test substance in the diet throughout the study (M-027300-03-1). The doses were selected based on a dose finding experiment (M-027080-01-2), which was conducted neither under GLP principles nor according to an OECD Guideline. There, 10 male and 10 female rats were fed with 0, 20, 100 or 500 ppm of the test substance in pelleted diet. Administration was continued for a three-week prepairing period and throughout the pairing, gestation and lactation periods. Pups of the F1 generation were reared for a further week on the respective test diet after weaning (from post partum Day 21). It was shown that at 500 ppm, P females had a reduced body weight gain during the preparing period and consequently decreased mean body weights during the following gestation and lactation periods, they also consumed less food than control animals during the lactation period. In the F1 generation, decreased mean body weight on day 0 post partum and throughout the lactation and rearing period were observed in the 500 ppm group as well as reduced food consumption during the rearing period (days 21 - 28 post partum). Therefore, the NOEL of the test substance was set to 100 ppm and the dose rationale for the main study was set to 100, 250 and 700 ppm.

In the main study, 105 days before cohousing of P0 animals, treatment started in 30 animals per sex per dose group. After a maximum of 22 days of cohousing, pregnant rats were separated from the males and were continued to be fed with the test substance mixed in the diet during gestation and lactation. Two weeks after weaning of the first litter (F1A), P0 animals were cohoused again for mating. Treatment with the test substance continued throughout mating. After the mating period, male parental animals (P0) were sacrificed and treatment for female rats continued during gestation and lactation of the second litter (F1B). F1A pubs were sacrificed on Day 21 post partum (after weaning), from F1B litters, 26 males and females were chosen for further mating of the F2 generation. These 52 animals formed the P1 generation and were fed in a prepairing period with the test substance in the same dose groups for 105 days. For mating, they were cohoused 1 male to 1 female for a maximum of 21 days (no siblings were paired). F2A pubs were sacrificed post weaning (at ca. 21 days of age) and P1 animals were paired again circa 10 days thereafter. Pubs of the F2B litter were reared until weaning and sacrificed thereafter. All P1 animals were killed 21 days post partum.

All animals were examined daily for clinical signs and morbidity. Body weight and food consumption of P0 and P1 animals was monitored at least weekly during the prepairing, gestation and lactation period. Specified organs of all parental animals (P0 and P1) and of one male and female pub of each litter after sacrifice on Day 21 post partum were weighed and tissues preserved for possible histopathological examination. All animals underwent external and internal gross examination at necropsy. From P1 animals, blood for hematological and clinical chemistry assessment was taken before necropsy.

Five non- treatment related deaths occurred during the study in P0 (3) and P1 (2) animals. There was no dose-response relationship and deaths occurred in all but the highest treatment group. Reduced food consumption was observed for P0 and P1 animals that received 700 ppm of the test substance during the prepairing period. For females, reduced food consumption was also observed at 100, 300 and 700 ppm during all gestation and lactation periods (P0 and P1 animals onyl at 700 ppm). Consequently, animals of the 700 ppm group had reduced body weight. The body weight gain was especially reduced during the prepairing period of P0 animals (male and female), after that, it was similar to controls but body weight remained low because of the established weight difference. In all offspring generations of the 700 ppm dose group (F1A, F1B, F2A and F2B), reduced body weight gain was observed. In the P1 generation, increased O-demethylase activity was observed for females receiving 250 ppm test substance and both sexes of the 700 ppm group. Also, N-demethylase activity and hepatic cytochrome P-450 content was increased in males at 700 ppm. This indicates increased xenobiotic metabolism but no further evidence of liver toxicity was found and it was therefore seen as a sign of adaptive processes. No teratogenic effects were found by external examination of the pups in any group of either generation. The NOAEL for parental and developmental toxicity were set at 250 ppm based on reduced body weight and feed consumption. No effects were seen on fertility up to the highest concentration, so that the NOAEL for reproduction was set at 700 ppm. 

 

The study was conducted under GLP guidelines and according to OECD Guideline 416 (1983). Deviations as compared to the current version of the guideline (2001) were as follows: the oestrus cycle as well as sperm parameters were not evaluated, pairs without progeny were not evaluated to determine cause of infertility, age of vaginal opening and preputial separation for F1 weanings selected for mating were not recorded, data on physical landmarks in pups and other postnatal developmental data were not recorded, uteri of primiparous females were not examined for presence of implantation sites, brains of parental animals and offspring were not weighed, spleen and thymus of offspring were not weighed.

References not included in IUC:

Detailed information on references not included in IUC are available in the CSR and in chapter 13.

Effects on developmental toxicity

Description of key information

Developmental toxicity (rat, OECD 414): 

NOAEL (maternal): 30 mg/kg bw/day

LOAEL (maternal): 100 mg/kg bw/day

NOAEL (developmental): 30 mg/kg bw/day

LOAEL (developmental): 100 mg/kg bw/day

NOAEL (teratogenicity): > 100 mg/kg bw/day

 

Developmental toxicity (rabbit, OECD 414): 

NOAEL (maternal): 24 mg/kg bw/day

LOAEL (maternal): 72 mg/kg bw/day

NOAEL (developmental): 24 mg/kg bw/day

LOAEL (developmental): 72 mg/kg bw/day

NOAEL (teratogenicity): > 72 mg/kg bw/day

 

 

2-generation (rat, OECD 416):

NOAEL (maternal, P0/F1): 250 ppm (correspondonding to ca. 20 mg/kg bw/day during premating period)

LOAEL (maternal, P0/F1): 700 ppm (correspondonding to ca. 50 mg/kg bw/day during premating period)

NOAEL (development, F1/F2): 250 ppm (correspondonding to ca. 40 mg/kg bw/day during lactation)

LOAEL (development, F1/F2): 700 ppm (correspondonding to ca. 100 mg/kg bw/day during lactation)

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Jul 1987 - 24 Nov 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 2018

Deviations:

yes

Remarks:

no weight and histopathology of thyroid gland; thyroid hormones not checked in blood, no gross pathology; treatment was terminated on Day 16 instead of one day prior to sacrifice (Day 20)

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 1984

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: KFM, Kleintierfarm Madoesrin AG, Fuellinsdorf, Switzerland
- Age at study initiation: 11 weeks, minimum
- Weight at study initiation: 184 - 240 g
- Housing: individually in Makrolon cages (type-3) with wire mesh tops and standardized granulated softwood bedding (Lignocel, Schill AG, Muttenz, Switzerland).
- Diet: Pelleted standard Kliba 343 rat/mouse maintenance diet ("Kliba", Klingentalmuehle AG, Kaiseraugst, Switzerland, Batch nos. 73/87 and 75/87), ad libitum
- Water: tap water, ad libitum
- Acclimation period: minimum 7 days prior to mating

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: 13 Jul To: 12 Aug 1987

Route of administration:

oral: gavage

Vehicle:

other:

Remarks:

distilled water with 0.5 % Cremophor EL

Mass median aerodynamic diameter (MMAD):

other: not applicable

Remarks on MMAD:

not applicable

Details on exposure:

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

VEHICLE
- name: distilled water with 0.5 % Cremophor EL
- Amount of vehicle: 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration, homogeneity and stability of the test article/vehicle mixtures were determined on one occasion before the dosing period. Samples were taken immediately after preparation and again 2 hours later. During the dosing period, samples were taken for confirmation of concentration and homogeneity on one occasion. Analysis was done using a HPLC.

It was shown that the test material was stable in the vehicle for up to two hours. The mean concentrations found were in the range of 81.9 % to 96.6 % of the nominal concentration. Homogenity was also confirmed.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: overnight
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: no
- Proof of pregnancy: sperm in vaginal smear referred to as Day 0 post coitum (Day 0 of pregnancy)

Duration of treatment / exposure:

from Day 6 through to Day 15

Frequency of treatment:

daily

Duration of test:

from Day 0 of pregnancy to Day 21

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosages were based on the results of the dose range-finding study (RCC Project 083507). In this study, effects on maternal animals were observed at 50, 100 and 150 mg/kg bw/day (all doses tested) shown as reduced food consumption and body weight gain. Developmental toxicity and teratogenicity was not observed up to the highest dose. Therefore, 10, 30 and 100 mg/kg bw/day of the test substance were used in the main study.
- Fasting period before blood sampling for (rat) dam thyroid hormones: not examined
- Time of day for (rat) dam blood sampling: not examined

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: No data
- Time schedule: not reported

BODY WEIGHT: Yes
- Time schedule for examinations: daily

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: all internal organs, with emphasis upon the uterus, uterine contents, position of fetuses in the uterus and number of corpora lutea

Ovaries and uterine content:

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

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations (visceral and brain): Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data
- Anogenital distance of all live rodent pups: no

Statistics:

For data about body weights, food consumption, reproduction and skeletal examination:

One-way analysis of variance (ANOVA) to test for significance between tretment groups
If a normal distribution could be assumed, a Dunnett's test was performed for the comparison between the treated groups and the control group. When no normal distribution was assumed, the Steel-test (many-one rank test) was applied. Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information.

Indices:

Pre implantation loss (%) was calculated as
(number of corpora lutea - number of implantations)/(number of corpora lutea) x 100

Post implantation loss (%) was calculated as
(number of implantations - number of live fetuses)/(number of implantations) x 100

sex ratio of males/females (%) was calculated as
(number of males/females)/(number of fetuses) x 100

Historical control data:

Yes, available in the study report

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

In one female of the control group and in one female of the high dose group, bald areas in the dorsal thoracic region were noted from days 15 and 11 post coitum, respectively. This is a common finding in pregnant animals of this strain and age.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality:

no mortality observed

Description (incidence):

not applicable

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 30 mg/kg bw/day: reduced body weight gain throughout the study (corrected bw gain of 5.6 g instead of 7.9 g in controls from Day 6 - Day 21 (non-adverse))
- 100 mg/kg bw/day: statistically significantly reduced body weight gain throughout the study (corrected bw gain of 4.2 g instead of 7.9 g in controls from Day 6 - Day 21)

Details can be found in Table 1 in Attachment 1.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- 10 mg/kg bw/day: reduced food consumption from Day 6-11 compared to controls (-9.5%), increased food consumption from Day 11-16 (+6.8% compared to controls ), the increase in food consumption was also found on Day 16-21 (post-treatment, +10.0% compared to controls )
- 30 mg/kg bw/day: reduced food consumption compared to controls from Day 6-11 (-10.0%)
- 100 mg/kg bw/day: reduced food consumption compared to controls from Day 6-11 (-9.5%) and from Day 11-16 (-19.9%), after the end of treatment (Day 16-21), food consumption was increased (+20.5%), all compared to controls.

Details can be found in Table 2 in Attachment 2.

Food efficiency:

not examined

Description (incidence and severity):

not applicable

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

not examined

Description (incidence and severity):

not applicable

Ophthalmological findings:

not examined

Description (incidence and severity):

not applicable

Haematological findings:

not examined

Description (incidence and severity):

not applicable

Clinical biochemistry findings:

not examined

Description (incidence and severity):

not applicable

Urinalysis findings:

not examined

Description (incidence and severity):

not applicable

Behaviour (functional findings):

not examined

Description (incidence and severity):

not applicable

Immunological findings:

not examined

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No abnormal findings were noted in any female of any group.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No abnormal findings were noted in any female of any group.

Neuropathological findings:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

not applicable

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

not applicable

Other effects:

not examined

Description (incidence and severity):

not applicable

Details on results:

Reproduction data

No test article-related or statistically significant differences between the mean values for the reproduction data of all dose-groups and the vehicle control group were noted. All differences observed were within the normal range of variation for animals of this strain and age.

Number of abortions:

no effects observed

Description (incidence and severity):

not applicable

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

not applicable

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

not applicable

Early or late resorptions:

no effects observed

Description (incidence and severity):

not applicable

Dead fetuses:

no effects observed

Description (incidence and severity):

not applicable

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

not applicable

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

not applicable

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: No adverse effects observed at this dose level.

Key result

Dose descriptor:

LOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

not applicable

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

not applicable

Changes in sex ratio:

effects observed, non-treatment-related

Description (incidence and severity):

-100 mg/kg bw/day: sex ratio was m/f 58.6/41.4% compared to 51.0/49.0% in controls. However, this was seen as incidental since In the dose range finding study (RCC Project 083507) to this study, the sex ratio of the high dose group was also shifted but in favour of the female fetuses. Further, in this dose range finding study, a sex ratio of 50:50 was noted at a higher dose level of 150 mg/kg bw/day.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

not applicable

Changes in postnatal survival:

not examined

Description (incidence and severity):

not applicable

External malformations:

no effects observed

Description (incidence and severity):

not applicable

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

- 100 mg/kg bw/day: wavy ribs were found more frequently, there were higher stages of skeletal ossification for example for both forelimbs, hind limbs, and lower ossification for example for cervical vertebrae and the sternum. As wavy ribes constitute a slight, transient alteration, which is fully reversed if pups are raised to age, the occurrence of wavy ribs is not rated as an adverse effect

Summarized Data can be found in Table 3 and 4 in Attachment 3 and 4 in the attached background material

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- 10 mg/kg bw/day: one fetus was noted with visceral malformations in form of severe haemorrhage around the brain and dilated lateral ventricles of the brain; the heart was enlarged - the auricles were dilated with blood and the walls of the ventricles were thickened. Since this was a single occurrence and was not found in higher doses, this finding was considered to be incidental and not related to treatment. The internal bleeding had caused increased body weight (7.5 grams cf. group mean of 4.8 grams) and was possibly the consequence of an injury.

Other effects:

not examined

Description (incidence and severity):

not applicable

Details on embryotoxic / teratogenic effects:

not reported

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed at this dose

Key result

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

Key result

Dose descriptor:

NOAEL

Remarks:

teratogenicty

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at the highest dose tested

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

skeletal: rib

Description (incidence and severity):

Wavy ribs, higher stages of skeletal ossification for example for both forelimbs, hind limbs, and lower ossification for example for cervical vertebrae and the sternum at the highest dose level of 100 mg/kg bw/day, regarded as secondary effects to maternal toxicity and therefore not considered directly treatment-related.

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

100 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:

no

Conclusions:

In the current study, developmental toxicity was evaluated in rats. Dams received 10, 30 or 100 mg/kg bw test substance per day via gavage (25 dams/dose) from Day 6 of pregnancy to Day 16. They were sacrificed on Day 21. Appearance, behaviour, and mortality of the dams were unchanged up to and including 100 mg/kg bw/day. A NOAEL for maternal toxicity of 30 mg/kg bw/day was derived based on initial body weight loss, reduced food consumption, and decreased body weight gain at 100 mg/kg bw/day (as discussed in the biocidal assessment report, Assessment Report, 2011). Fetuses of the control and treatment groups did not differ for external and visceral examination. Slight effects on skeletal development of fetuses, described as increased incidence of wavy ribs, were reported for the highest dose level of 100 mg/kg bw/day. However, these findings clearly correlated with obvious maternal toxicity. There was no evidence for teratogenicity in rat at doses up to including the highest tested level of 100 mg/kg bw/day.

The study was conducted under GLP conditions, according to according to OECD TG 414, adopted 1981. Therefore, examinations that are required by the current guideline, namely thyroid gland weight and histopathology were not performed, neither was blood analyzed for T4, T3 and TSH assessment. Nevertheless, the study is considered reliable and fully acceptable.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 Jun 1987 - 24 Nov 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 2018

Deviations:

yes

Remarks:

thyroid gland weight and histopathology were not performed, neither was blood analyzed for T4, T3 and TSH assessment, treatment was performed until Day 18 post coitum not until one day before sacrifice (Day 27)

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 1984

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

Chinchilla

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: KFM, Kleintierfarm Madoerin AG, Fuellinsdorf, Switzerland
- Age at study initiation: Between 4 and 6 months
- Weight at study initiation: 2650 - 4064 g
- Fasting period before study: not applicable
- Housing: individually in stainless steel cages equipped with an automatic cleaning system.
- Diet: Pelleted standard Kliba 341 rabbit maintenance diet ("Kliba", Klingentalmuehle AG, Kaiseraugst, Switzerland, Batch nos. 26/87, 27/87), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 7 days prior to mating

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 +/- 3
- Humidity (%): 0 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 01 Jun To: 08 Jul 1987

Route of administration:

oral: gavage

Vehicle:

other: distilled water with 0.5% Cremophor EL

Mass median aerodynamic diameter (MMAD):

other: not applicable

Remarks on MMAD:

not applicable

Details on exposure:

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

VEHICLE
- Name: distilled water with 0.5% Cremophor EL
- Amount of vehicle: 4 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration, homogeneity and stability of the test article/vehicle mixtures were determined on one occasion before the dosing period. Samples were taken immediately after preparation and again 2 hours later. During the dosing period, samples were taken for confirmation of concentration and homogeneity on one occasion. Analysis was done using a HPLC.
It was shown that the test material was stable in the vehicle for up to two hours. The mean concentrations found were in the range of 77.8 % to 94.1 % of the nominal concentration. Homogenity was also confirmed.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: until copulation had been observed
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: no
- Proof of pregnancy: the day of mating was referred to as day 0 post coitum

Duration of treatment / exposure:

from Day 6 through to Day 18 post coitum

Frequency of treatment:

daily

Duration of test:

from Day 0 to Day 28 post coitum

Dose / conc.:

8 mg/kg bw/day (nominal)

Dose / conc.:

24 mg/kg bw/day (nominal)

Dose / conc.:

72 mg/kg bw/day (nominal)

No. of animals per sex per dose:

16

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosages were based on the results of the dose range-finding study (RCC Project 083520). In this study, 4 mated females were fed daily orally by gavage from Day 6 through to Day 18 post coitum. The doses in the range finding experiment were 50, 100 and 150 mg/kg bw/day and the vehicle used was distilled water with 0.5 % Cremophor EL. Surviving females were sacrificed on Day 28 post coitum and fetuses were examined. In this pre-study, all females of the high dose group died, dose-related reduced mean food consumption during the treatment period was noted in groups of the low and middle dose, correlating with reduced mean body weight. The reproduction data showed a high post-implantation loss and as a consequence, a distinctly reduced mean number of live fetuses for dams receiving 100 mg/kg bw/day. Even though the numbe rof fetuses per litter were dose-related reduced in the low and middle dose group, no malformed or abnormal fetuses were found. It was concluded that 100 and 150 mg/kg bw/day caused distinct toxic effects in pregnant chinchilla rabbits and 50 mg/kg bw/day caused slight toxic effects. Therefore, dose levels of 8, 24 and 72 mg/kg body weight/day were chosen for the main study.
- Fasting period before blood sampling for dam thyroid hormones: no blood sampling done

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: daily from Day 0 until Day 28 post coitum

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- time schedule: recorded on Days 6, 11, 15, 19, 24 and 28 post coitum

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Gestation Day (GD) 28
- Organs examined: all internal organs, with emphasis on the uterus, uterine contents!, position of fetuses in the uterus and number of corpora lutea

Ovaries and uterine content:

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

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter
- Anogenital distance of all live rodent pups: not reported

Statistics:

For data about body weights, food consumption, reproduction and skeletal examination:
One-way analysis of variance (ANOVA) to test for significance between tretment groups
If a normal distribution could be assumed, a Dunnett's test was performed for the comparison between the treated groups and the control group. When no normal distribution was assumed, the Steeltest (many-one rank test) was applied. Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information.

Indices:

Pre implantation loss (%) was calculated as
(number of corpora lutea - number of implantations)/(number of corpora lutea) x 100

Post implantation loss (%) was calculated as
(number of implantations - number of live fetuses)/(number of implantations) x 100

sex ratio of males/females (%) was calculated as
(number of males/females)/(number of fetuses) x 100

Historical control data:

Yes, available in the study report

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- 72 mg/kg bw/day: 1 female had an abortion on Day 26 post coitum

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality:

mortality observed, treatment-related

Description (incidence):

- 72 mg/kg bw/day: 2 dams died on Day 18 and 19, one of the dead females showed white mucoid faeces during the three days prior to death, for both reduced food consumption and body weight compared to controls was recorded.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 24 mg/kg bw/day: body weight loss from the start of dosing (-0.9% weight gain from Day 6-11 compared to control animals) but with recovery during the dosing period, so that by termination on Day 28 post coitum, overall weight gain was similar to that of controls (-6% vs -5.9% corrected body weight gain in controls)
- 72 mg/kg bw/day: overall weight loss during the dosing period (weight gain of -5% compared to control animals), corrected body weight gain was more reduced compared to controls (-8.2% vs -5.9% in controls)

Summarized data can be found in Table 1 in Attachment 1.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- 24 mg/kg bw/day: reduction in mean food consumption compared to controls during the treatment period (-14.6% from Day 6-11)
- 72 mg/kg bw/day: reduction in mean food consumption compared to controls during the treatment period (-56.3% from Day 6-19), followed by increased food consumption during the post-treatment period (statisitcally signifant between Day 24-28 with +83.3%, but this was considered to be compensatory rather than treatment related).

Summarized data can be found in Table 2 in Attachment 2.

Food efficiency:

not examined

Description (incidence and severity):

not applicable

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

not examined

Description (incidence and severity):

not applicable

Ophthalmological findings:

not examined

Description (incidence and severity):

not applicable

Haematological findings:

not examined

Description (incidence and severity):

not applicable

Clinical biochemistry findings:

not examined

Description (incidence and severity):

not applicable

Urinalysis findings:

not examined

Description (incidence and severity):

not applicable

Behaviour (functional findings):

not examined

Description (incidence and severity):

not applicable

Immunological findings:

not examined

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

not applicable

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In each treatment group, one animal had slight indentations of the surface of the kidneys. Additionaly, one dam in the low dose group had a pale and slightly enlarged liver and in one of the high dose females which died, the abdominal cavity contained clear reddish fluid (ca. 500 mL).

Neuropathological findings:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

not applicable

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

not applicable

Description (incidence and severity):

not applicable

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

- 72 mg/kg bw/day: one female aborted on Day 26 post coitum

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

- 72 mg/kg bw/day: post-implantation loss was increased (10.8% when the dams that aborted or showed total resorption at necropsy were excluded and 32.5% when these animals were included in the calculation, compared to 4.2% in controls)

For summarized data see Table 3 (Attachment 3).

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

- 72 mg/kg bw/day: two females showed total resorption at necropsy on day 28 post coitum

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

- 72 mg/kg bw/day: in two dams, total resorption at necropsy on Day 28 post coitum was observed

For summarized data see Table 3 in the attached background material.

Dead fetuses:

no effects observed

Description (incidence and severity):

not applicable

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

not applicable

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

not applicable

Other effects:

not examined

Description (incidence and severity):

not applicable

Key result

Dose descriptor:

NOAEL

Effect level:

24 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed at this dose

Key result

Dose descriptor:

LOAEL

Effect level:

72 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

early or late resorptions

food consumption and compound intake

mortality

number of abortions

pre and post implantation loss

total litter losses by resorption

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

uterus

Description (incidence and severity):

Dams had early or late resoptions, higher number of abortions, pre and post implantation losses and higher total litter losses by resoption from 24 mg/kg bw/day on (considered secondary due to maternal systemic toxicity).

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 72 mg/kg bw/day: body weight was significantly reduced compared to controls (-9.7%)

In the 8 mg/kg bw/day group, the statistically significant reduction of 5.6% compared to controls was considered incidental since it was caused caused by the very low body weights of fetuses of a single dam/litter.

Details can be found in Table 3 (Attachment 3).

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

- 72 mg/kg bw/day: As a consequence of abortion and post-implantation losses, the number of live fetuses was decreased (83 compared to 136 in the control group)

For summarized data see Table 3 (Attachment 3).

Changes in sex ratio:

no effects observed

Description (incidence and severity):

not applicable

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

not applicable

Changes in postnatal survival:

not examined

Description (incidence and severity):

not applicable

External malformations:

effects observed, treatment-related

Description (incidence and severity):

- 72 mg/kg bw/day: body weights of fetuses were distinctly affected in 5 litters, and 3 fetuses (coming from 2 litters) were runts

In the 8 mg/kg/day bw group, 5/7 fetuses of 1 litter were runts and the other 2 had a reduced body weight of 20.2 and 21.6 g (compared to 32.3 g in the control). This finding was not considered treatment related but explained by body weight loss in the maternal animal of 291 grams between Days 14 and 23 post coitum. Thereafter, slight body weight gain was noted and at necropsy no abnormal findings were ascertained. The finding was not observed in other animals that reiceived the same dose and no signs of toxicity were observed, so that the finding can be considered incidental.

In head examinations, only one fetus of the 24 mg/kg bw/day group had hydrocephalus internus (both hemispheres). This finding was considered incidental and not treatment related.

Summarized data can be found in Table 3 (Attachment 3).

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Abnormal findings
Abnormal findings were observed but not considered treatment related. These were for example partially fused 6th and 7th left ribs; dumbbell shaped thoracic vertebral body no. 4 in 2 fetuses of the control group, occassional findings of abnormally ossified, missing, fused, asymmetric and/or bipartite sternebra in the middle and high dose group. For the high dose group this was considered to be the consequence of retarded maturation, indicated by the markedly reduced body weights in four of the five affected fetuses. All other findings were in the normal range of variations for this rabbit strain.

Summarized data can be found in Table 4 (Attachment 4).

Stage of development
- 72 mg/kg bw/day: compared to controls, increased number of non-ossified phalangeal nuclei and metacarpalia of both fore limbs and phalangeal nuclei of both hind limbs was found (caused by 3 litters with mean body weights of 25.5, 27.1 and 23.4 grams, group mean fetal body weight was 31.3 g). This finding was attributed to severe maternal toxicity.

Statsitical significant differences in one litter of the low-dose group were attributed to the weight loss of the dam as explained above and therefore not considered treatment related.

Summarized data can be found in Table 5 (Attachment 5).

Visceral malformations:

no effects observed

Description (incidence and severity):

not applicable

Details on embryotoxic / teratogenic effects:

No teratogenic effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

24 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at this dose

Key result

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

72 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

fetal/pup body weight changes

external malformations

skeletal malformations

Key result

Dose descriptor:

NOAEL

Remarks:

teratogenicity

Effect level:

72 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at the highest dose tested

Remarks on result:

other: no effects indicative of teratogenicity

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

other: see description

Description (incidence and severity):

Non-ossified phalangeal nuclei and metacarpalia of both fore limbs and phalangeal nuclei of both hind limbs at the highest dose level of 72 mg/kg bw/day, regarded as secondary effects to maternal toxicity and therefore not considered directly treatment-related.

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

72 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:

no

Conclusions:

In this study, pregnant rabbits were fed with the test material from Day 6 through Day 18 post coitum per gavage in doses of 8, 24 and 72 mg/kg bw/day. The test material was toxic to maternal animals at 72 mg/kg bw/day, as seen in mortality, distinctly reduced food consumption and body weight gain and a higher post-implantation loss in comparison to control and the other dose groups. Only slight effects on food consumption and body weight gain were observed in the middle dose group (24 mg/kg bw/day). Therefore, the maternal NOAEL was set at 24 mg/kg bw/day. In fetuses of the high dose group, slight effects on body weights and skeletal development were observed, in coherence with maternal toxicity. The NOAEL for development was set to 24 mg/kg bw/day.
There was no evidence for teratogenicity in rabbit at doses up to including the highest tested level of 72 mg/kg bw/day.

The study was conducted under GLP conditions, according to OECD TG 414, adopted 1981. Therefore, examinations that are required by the current guideline, namely thyroid gland weight and histopathology were not performed, neither was blood analyzed for T4, T3 and TSH assessment. Nevertheless, the study is considered reliable and fully acceptable.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

24 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

The developmental toxicity was assessed in rodent (rat) and non-rodent (rabbit), according to OECD TG 414 (1981), under GLP conditions. Both studies are considered of reliable quality and validity, fulfilling the criteria of a key study. Thus, both are suitable for assessment of the present endpoint.

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

To assess developmental toxicity, the effects of the test material were investigated in rats and rabbits.

Studies performed in rats

With respect to the key study conducted with rat, a range-finding preliminary study (M-026519-01-1, non GLP, non-guideline) first was conducted. In this preliminary study, groups of 5 mated female rats were fed daily via gavage with 0, 50, 100 or 150 mg/kg bw/day test substance in distilled water with 0.5 % Cremophor EL. This treatment was done from Day 6 through to Day 15 of gestation. Mortality and clinical signs were observed twice daily. Food consumption was recorded every five days and body weights were recorded daily from Day 0 to Day 21 post coitum. On Day 21, all dams were sacrificed and the dams and fetuses were examined. No mortality was observed. Food consumption, body weight gain and body weight gain were reduced starting at 50 mg/kg bw/day. Apart from that, no clinical signs were observed. Also, there was no effect on reproduction data, no malformed or abnormal fetus was observed. Fetuses did not differ in mean body weight. Based on the results of the range finder, the test doses for the main study were set as follows:10, 30 and 100 mg/kg bw/day. 

In the main developmental toxicity study with rats (M-027900-04-1), the dams received 10, 30 or 100 mg/kg bw test substance per day via gavage (25 dams/dose) from Day 6 of pregnancy to Day 16. Mortality and clinical signs were observed twice daily. Food consumption was calculated for Day 0-6, Day 11-16 and Day 16-21 and body weights were recorded daily from Day 0 to Day 21 post coitum. Dams were sacrificed on Day 21 and underwent gross macroscopic examination with emphasis on the uterus. The uterus was weighed and uterine contents, position of fetuses and number of corpora lutea were recorded. There was no mortality in the study and treatment-related clinical signs observed. In the all dose groups, maternal animals showed reduced food consumption and body weight gain was also reduced in the high dose group. Therefore, the NOAEL was set at 30 mg/kg bw/day for maternal toxicity (LOAEL was 100 mg/kg bw/day). Fetuses of the control and treatment groups did not differ for external and visceral examination. However, at the highest dose level of 100 mg/kg bw/day, an increased incidence of skeletal malformations, described as increased incidence of wavy ribs, was reported. As discussed in the biocidal assessment report (Assessment Report, 2011), wavy ribs constitute a slight, transient alteration, which is fully reversed if pups are raised to age. As a consequence, the occurrence of wavy ribs is not rated as an adverse effect, and the developmental NOAEL in the rat teratogenicity study in rats is set at 100 mg/kg bw/day.

The study was conducted under GLP conditions, according to OECD TG 414, adopted 1981.Therefore, examinations that are required by the current guideline, namely thyroid gland weight and histopathology were not performed, neither was blood analyzed for T4, T3 and TSH assessment. Nevertheless, the study is considered relaibale and fully acceptable. 

 

Studies performed in rabbits

With respect to the key study conducted with rabbit, a range-finding preliminary study (non GLP, non-guideline) was conducted (M-026507-01-1). In this study, groups of 4 mated females received 50, 100 or 200 mg/kg bw/day of the test substance via gavage from Day 6 through to Day 18 post coitum. The vehicle (distilled water with 0.5 % Cremophor EL) was administered to the control group. Mortality and clinical signs were observed twice daily. Food consumption was recorded on Day 6, 11, 15, 19, 24 and 28 and body weights were recorded daily from Day 0 to Day 28 post coitum. Surviving females were sacrificed on Day 28 post coitum and fetuses were examined. All females in the high dose group died, dose-related reduced mean food consumption during the treatment period was noted in groups of the low and middle dose, correlating with reduced mean body weight. The reproduction data showed a high post-implantation loss and as a consequence, a distinctly reduced mean number of live fetuses for dams receiving 100 mg/kg bw/day. Even though the fetuses were dose-related reduced in the low and middle dose group, no malformed or abnormal fetuses were found. It was concluded that 100 and 150 mg/kg bw/day caused distinct toxic effects in pregnant chinchilla rabbits and 50 mg/kg bw/day caused slight toxic effects. Based on the results of the range finder, the test doses for the main study were set as follows: 8, 24 and 72 mg/kg body weight/day.

The main study (M-027920-04-1) in female pregnant rabbits was conducted similarly to the dose finding experiment. Doses of 8, 24 and 72 mg/kg bw/day were administered from Day 6 through to Day 28 and 16 dams were used per group. Mortality and clinical signs were observed twice daily. Food consumption was recorded on Day 6, 11, 15, 19, 24 and 28 and body weights were recorded daily from Day 0 to Day 28 post coitum. Surviving females were sacrificed on Day 28 post coitum and fetuses were examined. The high dose of 72 mg/kg bw/day caused severe toxicity in dams as seen by two deaths, one abortions and two total resorptions at terminal necropsy. Food consumption was reduced during the treatment period (-56.3%) as well as body weight and body weight gain (-5%). Effects on food consumption and body weight were also seen in the middle dose group of 24 mg/kg bw/day. Here, food consumption was 14.6% lower during the treatment compared to controls and a loss of body weight was observed from the start of dosing (-0.9% weight gain from Day 6-11). This weight loss was reversible, so that by termination on day 28 post coitum, overall weight gain was similar to that of controls (-6% vs -5.9% corrected body weight gain in controls). At 72 mg/kg bw/day post-implantation loss was also increased, even when the dam that aborted and the maternal animals with total resorption were excluded from the calculation (+10.8%, +32.5% when they were included). Moreover, body weights of the fetuses of the high dose group were slightly reduced compared to the controls. Skeletal examinations showed similar types of abnormal findings compared to controls but with a slightly higher incidence. Non-ossified phalangeal nuclei and metacarpalia of the fore limbs and phalangeal nuclei of the hind limbs occurred also more often in fetuses of the 72 mg/kg bw/day group. These incidences were attributed to immaturity, since the affected fetuses also showed reduced body weight. The finding of one fetus in the middle dose group with hydrocephalus internus was considered to be incidental.

Taken together, the data showed severe maternal toxicity at 72 mg/kg bw/day that also affected the fetuses. In line with the biocidal assessment report (Assessment Report, 2011), the dose of 24 mg/kg bw/day (related to decreased body weight gain and food consumption) is not considered as dose which causes adverse effects on maternal animals and in utero development. Thus, a NOAEL for systemic maternal toxicity of 24 mg/kg bw/day is defined, based on body weight losses, decreased food consumption and mortality observed at the next higher concentration of 72 mg/kg bw/day. Further, as discussed in the biocidal assessment report (Assessment Report, 2011), at the same dose, one female aborted on day 26 post coitum, and two females showed total litter resorption at terminal necropsy. The body weights of the foetuses were slightly reduced and the incidence of foetuses with retarded ossification increased at 72 mg/kg bw/day. Because of the reduced litter size in this group, which would have resulted in increased foetal weights had there not been foetal toxicity, the reduced foetal weights and the skeletal changes are regarded as signs of foetal retardation and may have resulted from the severe maternal toxicity. No treatment-related malformations were observed. Both, the maternal and fetal NOAEL for developmental toxicity is set at 24 mg/kg bw/day. As no effects indicative for teratogenicity were detected, a NOAEL of 72 mg/kg bw/day was defined in regard to teratogenicity.

 

The study was conducted under GLP conditions and according to OECD TG 414 (1981). Therefore, examinations that are required by the current guideline, namely thyroid gland weight and histopathology were not performed, neither was blood analyzed for T4, T3 and TSH assessment. Nevertheless, the study is considered reliable and fully acceptable.

 

References not included in IUC:

Detailed information on references not included in IUC are available in the CSR and in chapter 13.

Mode of Action Analysis / Human Relevance Framework

No data available.

Justification for classification or non-classification

Based on reliable key data, the test substance warrants no classification and labelling regarding reproduction toxicity according to CLP Regulation (EC) No. 1272/2008.