N-[2,5-dichloro-4-(1,1,2,3,3,3-hexafluoropropoxy)-phenyl-aminocarbonyl]-2,6-difluorobenzamide

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

- Oral: NOAEL for systemic toxicity, toxicity to reproduction and developmental toxicity = 250 ppm (equivalent to mean dietary concentration of 19.73/22.03 and 21.48/22.87 (F0/F1) for males and females, respectively during premating, rat, OECD TG 416, Fritzgerald 1992

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 May 1990 to 13 May 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

1983

Qualifier:

according to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

Version / remarks:

1982

Qualifier:

according to guideline

Guideline:

EU Method B.35 (Two-Generation Reproduction Toxicity Test)

Qualifier:

according to guideline

Guideline:

other: Japanese MAFF

Version / remarks:

1985

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Tif: RAI f (SPF), hybrids of RII/l x RII/2

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P0) x 6 to 7 wks
- Housing: The animals were housed individually (not counting litters) in macrolon cages with wire mesh top. The bedding material was standarised granualted soft wood.
- Diet: Pelleted standard feed ad libitum
- Water: Tap water ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 3
- Air changes (per hr): 16
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 28 May 1990 to 13 May 1992

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Ground feed pellets were mixed with the appropriate quantity of test substance and repressed to pellets with steam.

DIET PREPARATION
- Rate of preparation of diet: every four weeks
- Storage temperature of food: room temperature

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of 19 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as Day 0 of pregnancy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and stability of test substance in feed mixtures were established before commencing the study. In order to permit verification of content, homogeneity and stability under actual conditions of administration, samples of the diet mixes were taken during their preparation. On each day of pellet preparation, samples were taken prior to pellet pressing from the top, middle and bottom of the mix container, for each dose mixture (to assess homogeneity and achieved concentrations). Samples of pellets from the first 5 ppm batch were also taken after storage for 4 weeks at room temperature, to assess stability. All samples were frozen at approximately -20°C until analysis. Samples for analysis were transported frozen in ice-boxes containing frozen C02 (dry ice) to the analytical laboratory, together with 500 g of diet and approximately 2.0 g of test substance.

Frequency of treatment:

Continuously

Details on study schedule:

The test substance was administered to parental (P0) animals prior to their mating (10 weeks), during the resultant pregnancies, and through the weaning of their F1 offspring. The test substance continued to be administered to selected F1 offspring during their growth into adulthood, mating, and production of an F2 generation, up until weaning of the F2 generation.

Dose / conc.:

5 ppm

Remarks:

Low dose: Equivalent to mean dietary concentration of 0.39/0.42, 0.43/0.45 (P0/F1) for males and females, respectively during premating and 0.4/0.4, 0.95/0.83 (P0/F1) mg/kw bw/day for females during gestation and lactation, respectively.

Dose / conc.:

25 ppm

Remarks:

Intermediate dose: Equivalent to mean dietary concentration of 1.98/2.18 and 2.09/2.26 (P0/F1) for males and females, respectively during premating and 1.9/1.76, 4.13/3.96 mg/kw bw/day (P0/F1) for females during gestation and lactation, respectively

Dose / conc.:

100 ppm

Remarks:

Medium-high dose: Equivalent to mean dietary concentration of 7.80/8.80 and 8.50/8.80 (P0/F1) for males and females, respectively during premating and 7.7/7.36 and 17.13/15.76 mg/kw bw/day (P0/F1) for females during gestation and lactation, respectively.

Dose / conc.:

250 ppm

Remarks:

High dose: Equivalent to mean dietary concentration of 19.73/22.03 and 21.48/22.87 (P0/F1) for males and females, respectively during premating and 19.3/17.56 and 40.13/35.3 mg/kw bw/day (P0/F1) for females during gestation and lactation, respectively.

No. of animals per sex per dose:

30

Control animals:

yes, plain diet

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule: At least once daily
- Cage side observations: Clinical signs

BODY WEIGHT:
- Time schedule for examinations: In males, weekly from the start of the premating period until necropsy. In females, weekly from the start of the premating period until mating and on days 0, 7, 14 and 21 postcoitum and days 0, 7, 14 and 21 postpartum and/or weekly until necropsy

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

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 9 or more pups/litter. 4/sex/litter as nearly as possible; excess pups were killed and discarded and subjected to gross necropsy.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
- Litter Size & Sex: number of viable and stillborn pups on Day 0 postpartum; external sex of viable pups
- Mortality: daily from Day 0 postpartum
- Clinical Signs: at least once daily (normally a.m.; repeated p.m. if signs observed)
- Body Weight: individual pup weights on days 0, 4, 7, 14, and 21 postpartum

- Eye Opening: Number of pups per litter with both eyes open; examined daily on days 14, 15 and 16 postpartum
- Pinna Unfolding: Number of pups per litter with both eyes examined daily on days 2 to 4 postpartum
- Surface Righting: Number of pups per litter with all four feet on the ground within 30 seconds of being placed on the back; tested daily on days 2 to 4 postpartum
- Auditory Canal Opening: Number of pups per litter with external auditory meatus open bilaterally: examined daily on days 10 to 12 postpartum
These indices were calculated as the mean age in days at criterion by litter, e.g. a litter with five pups reaching criterion on days 2, 3, 3, 4 and 4 has a mean age at criterion of 3.2 days. Pups not reaching criterion were excluded from the analyses.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: Males were necropsied after delivery of the litter last sired
- Maternal animals: Females were necropsied after weaning of their litters.

GROSS NECROPSY
- Gross necropsy consisted of the vagina, uterus, ovaries, testes, epididymis, seminal vesicles, prostate, pituitary, skin, mammary area, spleen, mesenteric lymph node, axillary lymph node, popliteal lymph node, sternum with bone marrow, femur with joint, skeletal muscle, trachea, lung, heart, aorta, submandibular salivary glands, liver, pancreas, esophagus, stomach, small intestine, large intestine, kidneys, urinary bladder, adrenals, thyroid with parathyroid, thymus, peripheral nerve, spinal cord, brain, eyes with optic nerves, orbital glands, extraorbital lacrimal glands, zymbal glands, muzzle and tongue

ORGAN WEIGHTS
- Organs weights were recorded for: Ovaries, testes, spleen, heart, liver, kidneys, adrenals, thymus and brain.

HISTOPATHOLOGY
- Full histopathological examination was performed on the following organs/tissues, or representative samples thereof, in all control and high dose P (=F0) and F1 animals selected for mating: Vagina, uterus, ovaries, mammary area, testes, epididymus, seminal vesicles, prostate, pituitary and other organ/tissues taken at necropsy with gross macropathology or weight changes.
- Organs/tissues with histopathological changes in the high dose group were then examined in all animals from the lower dose groups.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days post partum.
- A complete gross necropsy was conducted on all pups not selected for mating, including pups culled, found dead or killed moribund. Culled pups were killed by decapitation; all other pups were killed by carbon dioxide inhalation.

GROSS NECROPSY
- Gross necropsy consisted of a macroscopic pathological examination of the main organs of the thoracic and abdominal cavities, with special attention directed to the organs of the reproductive system.

Statistics:

Statistical analyses of continuous data are performed using the Analysis of Variance Procedure (ANOVA) [2] followed by Dunnett's t-Test in case of a significant result in the ANOVA. Categorical data are analysed using Chi-Square test followed by Fisher's Exact test in case of a
significant result in the Chi-Square test. Non-parametric data are analysed using the Kruskal-Wallis nonparametric analysis of variance test
followed by Mann-Whitney U-test in case of a significant result in the Kruskal-Wallis test

Reproductive indices:

Female Mating: No. of females positively mated as % of no. of females used for mating
Female Fertility (=Pregnancy Index): No. of females pregnant as % of no. of females mated
Male Mating: No. of males producing positive mating as % of no. of males used for mating
Male Fertility: No. of males producing pregnancy as % of no. of males producing mating
Parturition: No. of females with births as % of no. of females with confirmed pregnancy
Gestation: No. of females with liveborn as % of no. of females with confirmed pregnancy

Offspring viability indices:

Live Birth: Mean pups born alive per litter as % of mean pups born per litter
Viability: Mean pups alive on day 4 (pre-culling) per litter as % of mean pups born alive per litter
Lactation: Mean pups alive on day 21 per litter as % of mean pups alive on day 4 (post-culling) per litter (= Viability Index at Weaning) (= Weaning Index)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

One female in the 250 ppm group was electively sacrificed with vaginal prolapse on day 113 (during lactation).
Wounds and/or crust on back or neck (forelimb in male) was seen in all dose groups, at various times during treatment.
Chromodacryorrhea was seen in one male in the 250 ppm group on days 18 to 42, and in one female in the 25 ppm group on days 29 to 49.
None of the above signs was considered to be treatment-related.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no parental mortalities in the P generation. One female in the 250 ppm group was electively sacrificed with vaginal prolapse on day 113 (during lactation).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Absolute body weight was not affected. Body weight gain was unaffected by treatment (Mean body weight gain of females in the 100 and 250 ppm groups was higher than controls from day 64 to 68, during the premating period; because of its isolated occurrence, this was not considered to be a treatment effect).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Feed consumption was unaffected by treatment (Mean feed consumption during the premating period was lower than control values in the male 5 and 25 ppm groups from day 106 to 113, and in the female 100 ppm group from day 36 to 43; because of their isolated occurrence and lack of dose relationship, these were not considered to be treatment effects).

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Histopathological examination revealed no treatment-related pathological changes in parental male and female animals.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

- The number of pairs not mating was 3, 2, 0, 3, and 6 in the control to 250 ppm group, respectively. Of the females mated, 2, 1, 1, 1, and 1 were not pregnant in the control to 250 ppm group, respectively. Thus there was no effect of treatment on mating and fertility indices. The mean number of mating days until positive mating occurred was not affected by treatment.
- No females died before, during or after gestation. Two pregnant animals did not deliver one in the 25 ppm group and one in the 100 ppm group; both presumably had total resorptions, as they were pregnant by Salewski stain at necropsy but no signs of abortions or dead pups were seen. Gestation and parturition indices were therefore not affected by treatment. Duration of gestation was approximately 22 days in all groups, and was not affected by treatment. All dams delivered live young, and the number with stillborn pups did not differ between groups.
- Prenatal losses (number of implantation sites not producing pups) were about 10% per group and were not affected by treatment. Perinatal losses (stillbirths) were about 1% and were not affected by treatment.

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

> 250 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Equivalent to mean dietary concentration of 19.73 and 21.48 for males and females, respectively during premating

Key result

Dose descriptor:

NOAEL

Remarks:

Toxicity to reproduction

Effect level:

> 250 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Equivalent to mean dietary concentration of 19.73 and 21.48 for males and females, respectively during premating

Key result

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

- The incidence of wounds and/or crust on ear, neck, back or flank was considered to have been increased by treatment in the male 250 ppm group
- Chromodacryorrhea was seen in two males (5 ppm group and 100 ppm group), and in four females (in the control group and 100 ppm group); this was not considered to be treatment-related.
- One female in the 25 ppm group had perineal haemorrhagic discharge from day 15 after mating, and pilorection starting the next day, both continuing intermittently until day 26 after mating (this animal did not deliver, but was pregnant by SaleWSki stain at necropsy). This was not considered to be treatment-related.
- Palpable masses were seen in one female in the 100 ppm group and in two females in the 250 ppm group. One female had a mass on the right flank and two females had a mass on the forelimb (right and left, respectively), first seen on gestation day 21

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no parental mortalities

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- Body weights in the 250 ppm group (males and females) were significantly (up to 10%) higher than in controls from the start of dosing throughout the premating period. This must have been due to increased weight gain in these animals occurring after lactation, since body weights at lactation did not differ between groups. Female body weights during gestation and lactation were no longer significantly higher than control values. Mean body weights in the 100 ppm group (both males and females) were marginally but not significantly lower than control values.
- Body weight gain in 250 ppm males was significantly higher than controls from day 57 to 64 and 106 to 113, and in 250 ppm females from day 36 to 43 and 50 to 57. These effects may be secondary to the higher body weights in these animals. The 5 ppm female group had significantly lower body weight gain than controls from day 8 to 15, 43 to 50, 57 to 64, and overall (1 to 71), and a higher body weight gain than controls from day 50 to 57; the 25 ppm female group had significantly lower body weight gain than controls from day 57 to 64. These effects were not considered to be related to treatment, because of their scattered occurrence and lack of dose dependency.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption in 250 ppm males was significantly higher than in controls in seven (Day 1 to 8, Day 22 to 29, Day 29 to 36, Day 36 to 43, Day99 to 106, Day 106 to 113, and Day 113 to 120) of 14 intervals during the premating period. Female food consumption was not affected by treatment. Significantly lower feed consumption than controls was seen during the premating period in the female 5 and 100 ppm groups (days 1 to 8 and 36 to 43 in the 5 ppm females; days 1 to 15 in the 100 ppm females); because of their scattered occurrence and lack of dose dependency, these effects were not considered to be treatment- related.

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- The 250 ppm males had heavier whole body, spleen, heart, liver, kidneys, testes and adrenals than controls; relative brain weight was lower than in controls. These effects are considered to be secondary to the (treatment-related) heavier body weights in this group
- The 250 ppm females had heavier whole body, spleen, heart, and adrenals than controls; relative kidney and brain weights were lower than in controls. These effects are considered to be secondary to the (treatment-related) heavier body weights in this group. There were no statistical differences from control in the organ to body weight ratios in the 250 ppm group.
The following isolated differences from control values were not considered to be related to treatment:
- higher absolute (not relative) heart weight in 25 ppm males,
- lower absolute and relative liver weight in 100 ppm females,
- higher relative (not absolute) ovary weight in 5 ppm females,
- higher relative (not absolute) adrenal weight in 100 ppm females,
- higher relative (not absolute) brain weight in 5 ppm females.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopical examination revealed no treatment-related pathological changes in parental male and female animals.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Histopathological examination revealed no treatment-related pathological changes in parental male and female animals.

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

- The number of pairs not mating was 2, 0, 3, 1, and 5 in the control to 250 ppm group, respectively. Of the females mated, 2, 1, 2, 3, and 2 were not pregnant in the control to 250 ppm group, respectively. Thus there was no effect of treatment on mating and fertility indices. The mean number of mating days until positive mating occurred was not affected by treatment
- No females died before, during or after gestation. Three pregnant animals did not deliver in the 25 ppm group (with perineal hemorrhagic discharge from day 15 post-mating), and two females in the 250 ppm group. (All three were pregnant by Salewski stain at necropsy but no abortions or dead pups were seen.) These effects were not considered to be treatment-related. Gestation and parturition indices were therefore not affected by treatment. Duration of gestation was approximately 22 days in all groups, and was not affected by treatment. All but one dam delivered live young; one female in the 250 ppm group delivered only one pup which was found dead and classified as "uncertain" with respect to stillbirth. All other dams delivered live young, and the number with stillborn pups did not differ between groups.
- Prenatal losses (number of implantation sites not producing pups) were not adversely affected by treatment. Perinatal losses (stillbirths) were about 1% and were not affected by treatment.

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

> 250 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Equivalent to mean dietary concentration of 22.03 and 22.87 for males and females, respectively during premating

Key result

Dose descriptor:

NOAEL

Remarks:

Toxicity to reproduction

Effect level:

> 250 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Equivalent to mean dietary concentration of 22.03 and 22.87 for males and females, respectively during premating

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

One pup (100 ppm group) was sacrificed moribund on lactation day 13 after a handling injury (fell on the floor and was stepped on). One pup from the 25 ppm group was sacrificed moribund on lactation day 14; it was suffering from inanition due to overgrowth of the lower incisors. This was not considered to be related to treatment. With the exception of the dead, cannibalised, missing and sacrificed pups considered above, there were no other notable clinical signs.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The loss of pups up to day 4 was about 1-2%, and was not affected by treatment. Loss of pups between days 4 and 21 postpartum was about 1-30, except in the 250 ppm group (6%). This higher loss was due to the loss of one litter, which had to be killed on lactation day 12 when the mother was electively sacrificed with a vaginal prolapse. In summary, live birth, viability and lactation indices were not affected by treatment.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Litter weights were not affected by treatment.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In the 25 ppm group, one pup had a discolored nose, which was related to its moribund condition (elective sacrifice after inanition due to elongated lower incisors), and other pup had no right testis.
In the 100 ppm group, one pup had dextrocardia. In the 250 ppm group, one pup had a small mass on the left caput epididymis, and two pups had bilateral testicular hypoplasia. Two pups, from the 5 and 25 ppm groups, were cannibalised, and one in the 250 ppm group had gas in stomach and intestine. None of the above findings was considered to be related to treatment.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Pup Physical/Behavioural Landmarks: Pinna unfolding, auditory canal and eye opening occurred at about the same time in all litters, and were not affected by treatment. Surface righting was minimally delayed by about 0.2 days in the 250 ppm group relative to controls.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 250 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Equivalent to mean dietary concentration of 19.73 and 21.48 for males and females, respectively during premating

Key result

Critical effects observed:

no

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

One pup (25 ppm group) was electively sacrificed on day 18 of lactation with ataxia of the hindlimbs, closed eyes and hypoactivity; this was a handling error - the pup had a back injury from being caught in the cage top during cleaning. One pup (100 ppm group) had an abdominal wound and cannibalized tail on day 0 of lactation; on day 1 it was not suckled, and on day 2 missing. With the exception of dead, cannibalised, missing and sacrificed pups, there were no other notable clinical signs.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The loss of pups up to day 4 was about 1-3%, and was not affected by treatment. Loss of pups from day 4 to 21 postpartum was about 0-1 %, except in the 250 ppm group (5%). This higher loss was due to the loss of one litter (three pups missing on days 8, 9 and 13, one cannibalised on day 14, and the remaining survivor electively sacrificed on day 16 due to lack of weight gain), and was not considered to be treatment-related. In summary, live birth, viability and lactation indices were not affected by treatment

Body weight and weight changes:

no effects observed

Description (incidence and severity):

F2 litter weights were not affected by treatment.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In the 25 ppm group, one pup (elective sacrifice on lactation day 18) had a thoracic spinal cord injury consistent with having been injured during cage cleaning. In the control group, one pup had bilateral testicular hypoplasia. In the 5 ppm group, one pup had unilateral renal hypoplasia. In the 100 ppm group, one pup had renal hyperplasia and fluid in the abdominal cavity. With the exception of findings confirming in-life observations (no milk in stomach, cannibalisation), there were no other notable necropsy observations. None of the above findings was considered to be related to treatment.

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Pup Physical/Behavioural Landmarks: Pinna unfolding, auditory canal and eye opening occurred at about the same time in all litters, and were not affected by treatment. Surface righting was minimally delayed by about 0.4 days in the 250 ppm group, relative to controls. There was considered to be no biologically relevant effect in the 100 ppm group, since performance of the pups in this group was comparable to controls on day 3.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

> 250 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Equivalent to mean dietary concentration of 22.03 and 22.87 for males and females, respectively during premating

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Table 1. Overall mean dose received during the premating period (mg/kg bw/day) (males and females), and during gestation and lactation (females)

Generation/Sex	Dietary concentration of the test substance (ppm)
	5	25	100	250
Premating F0 Parents:Males                   Females       F1 Parents:Males                   Females	0.39 0.43 0.42 0.45	1.98 2.09 2.18 2.26	7.80 8.50 8.80 9.35	19.73 21.48 22.03 22.87
Gestation F0 Parents: females F1 Parents: females	0.4 0.4	1.9 1.76	7.7 7.36	19.3 17.56
Lactation F0 Parents: females F1 Parents: females	0.95 0.83	4.13 3.96	17.13 15.76	40.13 35.3

Table 2. Physical/Behavioural landmarks summary F1 litters – Mean age in days of pups reaching criterion

Group group name dietary dose level		Group 1. control 0 ppm	Group 2. low 5 ppm	Group 3. intermediate 25 ppm	Group 4. medium-high 100 ppm	Group 5. high 250 ppm
Eye opening	Mean	15.0d	15.2	15.1	15.3	15.2
	S.D.	0.5	0.5	0.6	0.5	0.4
	N	25	27	27	24	22
	p- value	0.324
	pups reaching criteria, %	100	99	98	95	100
Pinna unfolding	Mean	2.8d	2.9	2.8	2.9	2.9
	S.D.	0.4	0.4	0.5	0.4	0.3
	N	25	27	28	25	23
	p- value	0.591
	pups reaching criteria, %	100	100	100	100	100
Surface righting	Mean	2.2d	2.3	2.2	2.3	2.4**
	S.D.	0.2	0.3	0.2	0.2	0.3
	N	25	27	28	25	23
	p- value	0.003	0.351	1	0.18	0.007
	pups reaching criteria, %	100	100	100	100	100
Auditory canal opening	Mean	11.8d	11.9	11.9	11.9	11.9
	S.D.	0.3	0.2	0.2	0.3	0.3
	N	24	26	26	20	22
	p- value	0.395
	pups reaching criteria, %	87	77	80	62	86

Table 2. Physical/Behavioural landmarks summary F2 litters – Mean age in days of pups reaching criterio

Group group name dietary dose level		Group 1. control 0 ppm	Group 2. low 5 ppm	Group 3. intermediate 25 ppm	Group 4. medium-high 100 ppm	Group 5. high 250 ppm
Eye opening	Mean	14.9d	14.8	15.1	14.9	14.7
	S.D.	0.6	0.5	0.7	0.5	0.6
	N	26	29	24	26	20
	p- value	0.256
	pups reaching criteria, %	100	100	99	100	100
Pinna unfolding	Mean	2.8d	2.7	2.8	2.8	2.8
	S.D.	0.5	0.5	0.5	0.6	0.5
	N	26	29	24	26	20
	p- value	0.932
	pups reaching criteria, %	100	100	100	100	100
Surface righting	Mean	2.1d	2.1	2.2	2.2*	2.5**
	S.D.	0.2	0.1	0.2	0.2	0.3
	N	25	29	24	26	20
	p- value	0	0.993	0.83	0.022	0
	pups reaching criteria, %	100	100	100	100	99
Auditory canal opening	Mean	11.7 d	11.7	11.6	11.7	11.6
	S.D.	0.5	0.4	0.4	0.4	0.5
	N	24	28	22	24	19
	p- value	0.713
	pups reaching criteria, %	88	90	89	78	91

Conclusions:

Gonadal function, oestrus cycles, mating behavior, conception, parturition, lactation and weaning, and organ pathology, were not affected at doses up to and including 250 ppm. In the 250 ppm group, development of the righting reflex was minimally delayed in F1 and F2 pups, and body weights of F1 adults were increased. The NOAEL for systemic parental toxicity and reproduction was set at 250 ppm (equivalent to mean dietary concentration of 19.73/22.03 and 21.48/22.03 (F0/F1) for males and females, respectively during premating) The NOAEL for developmental toxicity was set at 250 ppm (equivalent to mean dietary concentration of 19.73/22.03 and 21.48/22.03 (F0/F1) for males and females, respectively during premating).

Executive summary:

In a 2-generation reproduction study, the test substance was administered to young virgin male and female adult Tif: RAI f (SPF), hybrids of RII/l x RII/2 rats according to OECD TG 416 and GLP principles. The rats were continuously exposed to the test substance admixed to feed at concentrations of 0, 5, 25, 100 or 250 ppm. Ten weeks after initiation of dietary exposure, they were paired 1:1 within each dose group (30 animals per sex and dose) until positive mating occurred or for 19 days, whichever came first. Dams were allowed to litter and suckle naturally. Litters were culled to 4 male and 4 female pups, where possible, on day 4 postpartum. Clinical signs, body weights, feed consumption, mating, gestation and delivery parameters, pup survival and development were recorded. A gross necropsy examination was performed on all pups not selected for mating. All parental animals were necropsied after weaning of the offspring and subjected to pathological examination, with histopathology of the sexual and target organs.

Results showed for the P generation that the mean nominal test substance intake in males was initially approximately 0, 1, 3, 10 and 30 mg/kg body weight/day; by the end of the premating period, it had reduced to approximately half these values. Intake in females was similar until the lactation period, when, due to increased food intake, it rose to approximately double these doses (0, 1, 5, 20 and 50 mg/kg body weight/day). There were no mortalities or treatment- related clinical observations. Body weights and feed consumption were unaffected by treatment. Male and female mating and fertility indices, maternal gestation and parturition indices, and duration of gestation, were unaffected by treatment. In F1 offspring, livebirth, postnatal survival indices and litter weights were unaffected by treatment. The development of physical landmarks (pinna unfolding, auditory canal and eye opening) was unaffected by treatment, but appearance of the surface righting reflex was minimally delayed (by about 0.2 days) in the 250 ppm group relative to controls. There were no treatment- related observations at gross necropsy in P animals or F1 offspring, no effects of treatment on organ weights, and no treatment-related histopathological changes in the reproductive system or in any other organs examined in the P adult animals. For the F1 generation, the mean nominal test substance intake in males was initially approximately 0, 1, 3, 10 and 30 mg/kg body weight/day; by the end of the premating period, it had reduced to approximately half these values. Intake in females was similar until the lactation period, when, due to increased food intake, it rose to approximately double these doses (0, l, 5, 20 and 50 mg/kg body weight/day. There were no mortalities in F1 adults. The incidence of skin wounds/crust on head/trunk was increased in the male 250 ppm group. Body weights in 250 ppm males and females were significantly higher than in controls during the premating period. Feed consumption was increased only in 250 ppm males during the premating period. Male and female mating and fertility indices, maternal gestation and parturition indices, and duration of gestation, were unaffected by treatment. In F2 offspring, livebirth, viability and lactation survival indices and litter weights were unaffected by treatment. The development of physical landmarks (pinna unfolding, auditory canal and eye opening) was unaffected by treatment, but appearance of the surface righting reflex was minimally delayed in the 250 ppm group by about 0.4 days relative to controls. There were no treatment- related observations at gross necropsy in F1 animals or F2 offspring. Organ weights were higher in 250 ppm males and females, consistent with the increased body weights in this group. There were no treatment- related histopathological changes in the reproductive system or in any other organs examined in the F1 adult animals.

In conclusion, gonadal function, oestrus cycles, mating behavior, conception, parturition, lactation and weaning, and organ pathology, were not affected at doses up to and including 250 ppm. In the 250 ppm group, development of the righting reflex was minimally delayed in F1 and F2 pups, and body weights of F1 adults were increased. The NOAEL for systemic parental toxicity and reproduction was set at 250 ppm (equivalent to mean dietary concentration of 19.73/22.03 and 21.48/22.03 (F0/F1) for males and females, respectively during premating) The NOAEL for developmental toxicity was set at 250 ppm (equivalent to mean dietary concentration of 19.73/22.03 and 21.48/22.03 (F0/F1) for males and females, respectively during premating).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

19.73 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Guideline study OECD TG 416 performed in compliance with GLP

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

An one-generation study for a metabolite of the test substance is available the NOAEL for systemic toxicity for parental animals was placed on 7.1 mg/kg bw/day because of a decreased body weight, increased liver weight. The NOAEL for pup development was placed at 7.1 mg/kg bw/day because of reduced body weight gain. The NOAEL for toxicity to reproduction was placed at 29.4 mg/kg bw/day because of a reduction in the number of implantation sites and reduction in average litter size. In addition, delayed growth and delayed eye opening were observed.

Oral: 2-generation reproduction study, Fritzgerald 1992

In a 2-generation reproduction study, the test substance was administered to young virgin male and female adult Tif: RAI f (SPF), hybrids of RII/l x RII/2 rats according to OECD TG 416 and GLP principles. The rats were continuously exposed to the test substance admixed to feed at concentrations of 0, 5, 25, 100 or 250 ppm. Ten weeks after initiation of dietary exposure, they were paired 1:1 within each dose group (30 animals per sex and dose) until positive mating occurred or for 19 days, whichever came first. Dams were allowed to litter and suckle naturally. Litters were culled to 4 male and 4 female pups, where possible, on day 4 postpartum. Clinical signs, body weights, feed consumption, mating, gestation and delivery parameters, pup survival and development were recorded. A gross necropsy examination was performed on all pups not selected for mating. All parental animals were necropsied after weaning of the offspring and subjected to pathological examination, with histopathology of the sexual and target organs.

Results showed for the P generation that the mean nominal test substance intake in males was initially approximately 0, 1, 3, 10 and 30 mg/kg body weight/day; by the end of the premating period, it had reduced to approximately half these values. Intake in females was similar until the lactation period, when, due to increased food intake, it rose to approximately double these doses (0, 1, 5, 20 and 50 mg/kg body weight/day). There were no mortalities or treatment- related clinical observations. Body weights and feed consumption were unaffected by treatment. Male and female mating and fertility indices, maternal gestation and parturition indices, and duration of gestation, were unaffected by treatment. In F1 offspring, livebirth, postnatal survival indices and litter weights were unaffected by treatment. The development of physical landmarks (pinna unfolding, auditory canal and eye opening) was unaffected by treatment, but appearance of the surface righting reflex was minimally delayed (by about 0.2 days) in the 250 ppm group relative to controls. There were no treatment- related observations at gross necropsy in P animals or F1 offspring, no effects of treatment on organ weights, and no treatment-related histopathological changes in the reproductive system or in any other organs examined in the P adult animals. For the F1 generation, the mean nominal test substance intake in males was initially approximately 0, 1, 3, 10 and 30 mg/kg body weight/day; by the end of the premating period, it had reduced to approximately half these values. Intake in females was similar until the lactation period, when, due to increased food intake, it rose to approximately double these doses (0, l, 5, 20 and 50 mg/kg body weight/day. There were no mortalities in F1 adults. The incidence of skin wounds/crust on head/trunk was increased in the male 250 ppm group. Body weights in 250 ppm males and females were significantly higher than in controls during the premating period. Feed consumption was increased only in 250 ppm males during the premating period. Male and female mating and fertility indices, maternal gestation and parturition indices, and duration of gestation, were unaffected by treatment. In F2 offspring, livebirth, viability and lactation survival indices and litter weights were unaffected by treatment. The development of physical landmarks (pinna unfolding, auditory canal and eye opening) was unaffected by treatment, but appearance of the surface righting reflex was minimally delayed in the 250 ppm group by about 0.4 days relative to controls. There were no treatment- related observations at gross necropsy in F1 animals or F2 offspring. Organ weights were higher in 250 ppm males and females, consistent with the increased body weights in this group. There were no treatment- related histopathological changes in the reproductive system or in any other organs examined in the F1 adult animals.

In conclusion,gonadal function, oestrus cycles, mating behavior, conception, parturition, lactation and weaning, and organ pathology, were not affected at doses up to and including 250 ppm. In the 250 ppm group, development of the righting reflex was minimally delayed in F1 and F2 pups, and body weights of F1 adults were increased. The NOAEL for systemic parental toxicity and reproduction was set at 250 ppm (equivalent to mean dietary concentration of 19.73/22.03 and 21.48/22.03 (F0/F1) for males and females, respectively during premating) The NOAEL for developmental toxicity was set at 250 ppm (equivalent to mean dietary concentration of 19.73/22.03 and 21.48/22.03 (F0/F1) for males and females, respectively during premating)

Effects on developmental toxicity

Description of key information

- Oral: NOAEL for maternal toxicity is 500 mg/kg bw/day, the NOAEL for developmental toxicity is 1000 mg/kg bw/day, rat, OECD TG 414, Gilles 1989

- Oral: NOAEL for maternal toxicity and developmental toxicity is 1000 mg/kg bw/day, rabbit, OECD TG 414, Meyer 1989

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Jan 1989 to 7 Feb 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

1981

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

1988

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl: CD® (SD) BRVAF/Plus(tm) rats

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approx. 10 weeks of age at start of mating
- Housing: Rats were housed in hanging polycarbonate cages with solid bottoms containing approximately a one inch layer of hardwood chip bedding. Animals were housed in cages of approximately 62 sq. in. floor space prior to and after mating and approximately 143 sq. in. floor space during mating. Animals were caged individually except at the time of mating.
- Diet: Ad libitum, rodent chow
- Water: Ad libitum, tap water
- Acclimation period: at least 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-60
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 23 Jan 1989 to 26 Oct 1989

Route of administration:

oral: gavage

Vehicle:

other: Aqueous 3% cornstarch and 0.5% Tween 80

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was mixed with an aqueous 3% cornstarch and 0.5% Tween 80. The dose suspensions were prepared weekly by a weight/volume method and stored refrigerated.

VEHICLE
- Amount of vehicle: 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of all dose suspension preparations were taken for analytical determination of concentration and homogeneity. Analyses indicated that the mean measured concentrations of the test substance were within 10% of the intended concentrations for all preparations of the 20, 100 and 200 mg/mL suspensions. Analyses of dose suspensions for concentrations was based on six samples per suspension. The relative standard deviations ranged from 1.4 to 7.9% indicating adequate homogeneity.

Details on mating procedure:

- Impregnation procedure: Cohoused
- If cohoused: One or two females with each proven male breeder
- Length of cohabitation: Overnight
- Proof of pregnancy: Presence of sperm or a copulatory plug referred to as day 0 of pregnancy

Duration of treatment / exposure:

Day 6 through 15 p.c.

Frequency of treatment:

Once daily

Duration of test:

Untill Day 21 p.c.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule: Daily

BODY WEIGHT:
- Time schedule for examinations: Day 0, 6, 7, 9, 12, 16 and 21 of gestation

FOOD CONSUMPTION
- Time schedule for examinations: Day intervals 0-6, 6-9, 9-12, 12-16 and 16-21 of gestation

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day 21
- Organs examined: The oral cavity and all organs of the thoracic and abdominal cavities. The ovaries and uterus with cervix were immediately removed, trimmed, weighed intact and examined

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
- Soft tissue examinations: Yes
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Statistics:

Incidence data for each dose group, such as clinical observations were analysed by the Fisher exact probability test (Siegel, 1956) with Bonferroni’s correction for multiple comparisons to a single control level (Ingelfinger et al., 1983). Enumerative data for each litter, such as the percent incidence of foetal variations and malformations and the number of corpora lutea per dam, were analysed by the nonparametric Mann-Whitney U two-sample rank test (Goldstein, 1967). Quantitative or continuous data, such as body and food consumption were analysed by one-way analysis of variance (Steel and Torrie, l960) and the Dunnett’s t-test (Dunnett, 1964). The level of significance selected for all statistical tests was p < 0.05 but values also significant at p < 0.01 are indicated

Clinical signs:

effects observed, non-treatment-related

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No statistically significant reductions in mean absolute body weights or mean corrected body weights occurred at any dose level. The gain in body weight at 100 mg/kg bw/day was statistically reduced before, but not after, the start of treatment. The change in corrected body weight at 100 mg/kg bw/day was also statistically reduced for gravid days 0-21 but not from the start of treatment, gravid days 6-21. The statistically significant changes in mean body weights at 100 mg/kg/day were not considered treatment-related. The mean gain in body weight was reduced at 1000 mg/kg/ day after one day of treatment and was statistically reduced for the gravid day interval 7-9.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A statistically significant reduction in food consumption occurred over gravid days 6-9 at 1000 mg/kg/day.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant reductions in the mean number of implants/dam at 100 mg/kg/day were not considered to be adverse.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

General toxicity

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Dose descriptor:

NOAEL

Remarks:

Maternal developmental toxicity

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

The mean number of live foetuses per dam was slightly but statistically reduced at 100 and 1000 mg/kg/day but considered to be non-treatment related

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Malformations were observed in 19 foetuses. All of these malformations were considered to have occurred spontaneously. At 0 mg/kg/day, 6 foetuses from 5 litters were considered to be malformed. The most severely affected was a foetus (202/17 F; litter/foetus-sex) with absence of a lumbar vertebral centrum, absence of lumbar vertebrae and sacral vertebrae, and absence of the tail. Two foetuses from two litters had absence of a lumbar vertebra (213/2 F; 214/7 F). Two foetuses from one litter (223/14 and 16 F) had moderate dilation of a kidney pelvis. The sixth foetus (212/8 F) had moderate dilation of a kidney pelvis and extreme dilation of an ureter. At 100 mg/kg/day, six foetuses from four litters were considered malformed. The most severely affected was a foetus (258/4 F) that had incomplete situs inversus of the abdomen and thorax with only one lung lobe on the left side, moderate dilation of both kidney pelvis, and extreme convolution of both ureters. A second foetus in the same litter (258/16 M) had moderate dilation of both kidney pelvis. One foetus (254/5 F) had no anus and a rudimentary tail. One foetus from litter 257 (12 F) had umbilical eventration. A second foetus from litter 257 (8 M) had moderate dilation of a kidney pelvis. The sixth foetus (250/15M) had umbilical eventration. At 500 mg/kg/day, three foetuses from two litters were considered malformed. One foetus (283/2 H) had an absence of a lumbar vertebra. In litter 286 one foetus (1 F) had moderate dilation of both kidney pelvis and one foetus (9 F) had moderate dilation of one kidney pelvis. At 1000 mg/kg bw/day four foetuses from four litters were considered malformed. One foetus (322/3 F) had situs inversus of the abdomen and thorax, two foetuses (325/11 F; 335/1 F) had extreme dilation and convolution of both ureters and a fourth foetus (342/4 F) had moderate dilation of a kidney pelvis

Visceral malformations:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

The reduction at 100 mg/kg/day reflected a slight reduction in the number of corpora lutea (developed before treatment) and at 1000 mg/kg/day the <4% reduction in mean number of live foetuses compared to concurrent controls exceeded the mean values for controls from the previous five studies.

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Analytical verification:

Table 1. Analysis of the dosing suspensions

Date prepared and analysed	Concentration expected (mg/mL)	Number of samples	Mean concentration (mg/mL)	Range (mg/mL)	Standard deviation	Relative standard deviation (%)
1-20-89	20	6	18.4	16.9-19.6	1.3	7.0
	100	6	97	93-101	3	3.1
	200	6	198	189-205	5	2.7
1-27-89	20	6	18.1	16.0-19.9	1.4	7.9
	100	6	98	93-104	5	4.7
	200	6	194	166-206	14	7.4
2-3-89	20	6	18.5	17.5-19.2	0.7	3.9
	100	6		89-102	5	5.2
	200	6	202	199-207	3	1.4

Table 2. Intergroup comparison of maternal body weight gain (g)

Days	Dose level of mg test substance technical/kg/day
	0	100	500	1000
7-9	11	9.4	13	3.4*
Number#	22	23	19	25

#Number of females with live fetuses in utero at termination.

*Statistically significant difference from control group mean, p<0.05 (Dunnett’s t-test)

Table 3. Intergroup comparison of maternal food consumption (g/day)

Interval (days)	Dose level of mg test substace technical/kg/day
	0	100	500	1000
6-9	24	23	24	21*
Number#	22	23	19	25

#Number of females with live fetuses in utero at termination.

*Statistically significant difference from control group mean, p<0.05 (Dunnett’s t-test)

Table 4. Intergroup comparison of selected intrauterine data

Parameter	Dose level of mg test substance technical/kg/day
	0	100	500	1000
Mean number of implants per dam	16.5	15.4*	16.4	16.2
Mean number of implants per dam	15.9	15.0*	15.7	15.3*

*Statistically significant difference from control group mean, p<0.05 (two-tailed)

Table 5. Historical control data of selected intrauterine data

Parameter	Study number (start date)
	12 (11/4/88)	11 (22/6/87)	10 (16/3/87)	9 (22/7/85)	8a(14/5/85)
Mean number of implants per dam	15.8	14.4	14.2	14.7	15.6
Mean number of live fetuses per dam	15.0	13.8	12.8	14.2	14.2

Table 6. Intrauterine data for gravid intergroup comparison of maternal food consumption (g/day).

	Dose level of mg test substance technical/kg/day
	0		100		500		1000
Dams with implants	22		23		19		25
Dams with live fetuses	22		23		19		25
Dams with resorptions	10		8		10		17
Dams with affected implants^a	14		11		12		17
	Mean^b	SD	Mean	SD	Mean	SD	Mean	SD
Corpora lutea/dam Implants/dam	17.5 16.5	3.2 2.6	16.5 15.4*	1.9 1.9	18.5 16.4	3.1 2.6	18.1 16.2	3.1 1.3
Implantation index^c(%) Pre implantation loss^d(%)	95.3 4.7	13.1 13.1	93.8 6.2	10.2 10.2	89.3 10.7	12.0 12.0	91.1 8.9	11.6 11.6
Implant viability index^e(%) Post implantation loss^f(%)	95.7 4.3	8.1 8.1	97.5 2.5	3.7 3.7	95.8 4.2	4.9 4.9	94.4 5.6	5.3 5.3
Live fetuses/dam Dead fetuses/dam	15.9 0.0	2.9 0.0	15.0* 0.0	1.8 0.0	15.7 0.0	2.6 0.0	15.3* 0.0	1.4 0.0
Resorptions/dam - early - mid - late	0.5 0.1 0.0	0.6 0.5 0.0	0.3 0.1 0.0	0.5 0.3 0.0	0.3 0.2 0.2	0.5 0.4 0.5	0.5 0.4 0.0	0.7 0.7 0.0
Malformed live foetuses^g(%) Affected implants^h(%)	1.6 5.9	3.3 8.0	1.7 4.1	3.9 4.9	1.1 5.3	3.9 5.1	1.1 6.6	2.6 6.3
Sex ratio (%)^i	50.2	15.2	45.7	12.4	45.6	13.1	49.3	12.2
Mean body weights (grams)/live foetus Reproductive tract weight (grams)/dam	5.2 108.03	0.2 18.0	5.4 105.61	0.4 11.25	5.2 107.66	0.3 17.79	5.4 108.00	0.3 10.70

a Affected implants includes dead fetuses, resorptions and malformed live fetuses

b Values determined on a per litter basis

c (implants/corpora lutea)x 100

d 100 – implants index

e (viable implants/total implants) x 100

f 100 – implant viability index

g (fetuses with observations classified as malformations/total live fetuses) x 100

h [ (dead fetuses + resorptions + malformed live fetuses) /total implants] x 100

i (live female fetuses/total live fetuses) x 100

* Significantly different from 0 mg/kg /day dose level, p< 0.05, two-tailed

Conclusions:

Minimal maternal toxicity was evident at 1000 mg/kg bw/day and consisted of transient reductions in body weight gained and food consumed. No embryofoetal toxicity or teratogenic effects were apparent at any of the dose levels tested. The NOAEL in this study for maternal toxicity was 500 mg/kg bw/day and for developmental toxicity was 1000 mg/kg bw/day.

Executive summary:

The test substance was tested for embryotoxic and teratogenic action in accordance with OECD 414 and performed under GLP principles. The test substance was administered to Crl: CD® (SD) BRVAF/Plus™ rats by oral gavage as a suspension in aqueous 3% corn starch and 0.5% Tween 80 to groups of 25 mated females at dosages of 0, 100, 500 or 1000 mg/kg/day. The females were treated for 10 consecutive days from gestation day 6 through 15. The dose was based on each female’s gravid day 6 body weight. The females were monitored daily for clinical signs. Body weights and food consumption were recorded for selected intervals during gestation. The dams were sacrificed on gravid day 21; the status of the reproductive tract determined; and the foetuses examined for external, soft and tissue skeletal variations and malformations.

Results showed that treatment with the test substance caused minimal maternal toxicity at 1000 mg/kg/day indicated by a short period (gravid days 6-9) of slightly reduced body weight gain and food consumption. No embryofoetal and teratogenic toxicity was apparent at any dose level.

In conclusion, minimal maternal toxicity was evident at 1000 mg test substance/kg/day and consisted of transient reductions in body weight gained and food consumed. No embryofoetal toxicity or teratogenic effects were apparent at any of the dose levels tested. The NOAEL in this study for maternal toxicity was 500 mg/kg bw/day and for developmental toxicity was 1000 mg/kg bw/day.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Feb 1989 to 17 Mar 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

1981

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

1988

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Remarks:

Hra:NZW (SPF)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Females, approx. 6 months at the start of mating and males at least 5 months old
- Housing: Rabbits were housed in individual stainless steel cages with dimensions of 24" x 24" with grill bottoms. Clean cages were supplied weekly and clean pads at least twice a week
- Diet: Controlled amounts of food (for females, 125 grams/day prior to mating and 150 grams/day after mating and for males, 100 grams/day of Rabbit Chow
- Water: Ad libitum, tap water
- Acclimation period: At least 26 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17- 23
- Humidity (%): 40-60
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 13 Feb 1989 to 17 Mar 1989

Route of administration:

oral: gavage

Vehicle:

other: Aqueous 3% cornstarch and 0.5% Tween 80

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing preparations were made fresh weekly by a weight/volume method and stored refrigerated. Intended concentrations in mg/mL were 0, 20, 100 and 200.

VEHICLE
- Amount of vehicle: 5 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples from each batch of dosing preparation made were taken for analytical determination of concentration and homogeneity. Each analysis for concentration was based on six samples. Mean measured concentrations ranged from 92 to 105% of the intended concentrations. Relative standard deviations ranged from 2.0 to 9.8% indicating adequate homogeneity.
The test substance in 3% cornstarch and 0.5% Tween 80 at 21.9 mg/mL showed a 4.6% loss in concentration after 14 days stored refrigerated (4 °C) and warmed to room temperature prior to sampling. Therefore, dosing preparations made weekly were considered stable for the duration of use during the study.

Details on mating procedure:

- Impregnation procedure: Cohoused
- M/F ratio per cage: 1:1
- Proof of pregnancy: When mating was observed visually and succesful it is referred to Day 0 of pregnancy

Duration of treatment / exposure:

Days 7 through 19 p.c.

Frequency of treatment:

Once daily

Duration of test:

Untill Day 29 p.c.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

16

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected on the basis of a range finding teratology probe
- Rationale for animal assignment: The rabbit was selected as the species for this study based upon its wide acceptance by regulatory agencies for teratology studies.
The New Zealand White strain was chosen because of extensive usage and the availability of published background data for the incidence of spontaneous malformations.

Maternal examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule: Daily

BODY WEIGHT:
- Time schedule for examinations: On gravid days 0, 3, 7, 10, 13, 19, 24 and 29

FOOD CONSUMPTION
- Time schedule for examinations: Daily for gravid days 0 through 29.

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day 29
- Organs examined: All organs of the thoracic and abdominal cavities. The ovaries and uteri with cervices were immediately removed, trimmed of excess fat, weighed intact. Ovaries were weighed as a pair and placentas were weighed collectively for live fetuses. Each fetus was weighed. After fetuses, resorptions and placentas were removed, the uteri with cervices were weighed.

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
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: Yes

Statistics:

Incidence data for each dose group, such as clinical observations, were analysed by the Fisher exact probability test (Siegel, 1956) with Bonferroni’s correction for multiple comparisons to a single control level (Ingelfinger, et al., 1983). Enumerative data for each litter, such as the percent incidence of foetal anomalies and the number of corpora lutea per doe, were analysed by the nonparametric Mann-Whitney U two-sample rank test (Goldstein, 1967). Quantitative or continuous data, such as body weights and food consumption, were analysed by one-way analysis of variance (Steel and Torrie, 1960) and the Dunnett’s t-test (Dunnett, 1964). The level of significance selected for all statistical tests was p < 0.05, but values also significant at p < 0.01 are indicated.

Clinical signs:

effects observed, non-treatment-related

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female in the control group died on day 18 and one female in the 100 mg/kg/day group was found dead on day 11. Both of these deaths were a result of trauma induced by gavage dosing.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The fertility index (% of animals in a dose group that have conceptuses) was 88, 100, 75 and 94% for females at the 0, 100, 500 and 1000 mg/kg/day dose levels, respectively. There was no statistically significant treatment-related effect on fertility. All surviving pregnant females had live foetuses on gravid day 29.

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

General toxicity

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Remarks:

Maternal developmental toxicity

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One foetus (0 mg/kg bw/day) was observerd to have displaced thoracic vertebral centra, one foetus had an absent right rib and at 1000 mg/kg bw/day one foetus had fused ribs, one foetus had an absent left rib, one foetus had anophthalmia/microphtalmia, one foetus had branched ribs and horatic hemi-vertebrae.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The gall bladder was absent in one foetus (100 mg/kg bw/day)

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Results dosing preparation concentration and stability:

Each anlaysis was based on six samples. Mean measured concentrations ranged from 92 to 105% of the intended concentrations. Relative standard deviations ranged from 2.0 to 9.8%, indicating adequate homogeneity.

Table 1. Analysis of dose suspensions

			Intended test substance concentration (mg/mL)
			20			100			200
Date prepared	Analysis date	Batch	Mean	S.D.	R.S.D	Mean	S.D.	R.S.D	Mean	S.D	R.S.D
2/9/89	2/9/89	A	18.4	1.4	7.5	92	5	4.9	193	5	3.1
2/9/89	2/9/89	B	18.5	0.4	2.3	104	4	3.7	200	7	3.4
2/17/89	2/17/89	A	20.0	1.5	7.7	93	9	9.1	197	5	2.7
2/17/89	2/17/89	B	19.7	0.9	4.4	98.1	2.3	2.4	203	17	8.4
2/17/89	2/17/89	C	19.3	1.0	4.9	99	10	9.8	195	13	5.5
2/24/89	2/24/89	A	18.7	1.7	9.2	98.5	2.0	2.0	200	9	4.7
2/24/89	2/24/89	B	19.2	1.2	6.1	96.5	2.3	2.3	195	9	4.7
2/24/89C	2/27/89	A	19.8	1.3	6.4	105	7	7.1	206	11	5.3

Table 2. Summary of fertility and mortality

		Dose level (mg/kg bw/day)
		0	100	500	1000
Females	Assigned Treated	16 16	16 16	16 16	16 16
Not pregnant (%)		2(13)	0 (0)	4 (25)^a	1 (6)
Surviving (%)		2 (100)	0 (0)	4 (100)	1 (100)
Pregnant^b		14 (88)	16 (100)	12 (75)	15 (94)
Surviving (%)		13 (93)	15 (94)	12 (100)	15 (100)
Totally resorbed (%)		0 (0)	0 (0)	0 (0)	0 (0)
Dams with live foetuses on gravid day 21 (%)		13 (100)	15 (100)	12 (100)	15 (100)
(%) Aborted		0 (0)	0 (0)	0 (0)	0 (0)

a Female (56) was considered not pregnant since implant sites were observed upon examination of the uterus.

However, three corpora lutea were observed on the left ovary.

b Fertility index: the percentage of animals in a dose group that have conceptuses

Conclusions:

No maternal toxicity, embryofoetal toxicity or teratogenicity was observed at any of the dose levels tested. The NOAEL for maternal toxicity and developmental toxicity in this study was 1000 mg/kg bw/day.

Executive summary:

The test substance was tested for embryotoxic and teratogenic action in accordance with OECD 414 and performed under GLP principles. The test substance was administered by oral gavage to 16 mated female Hra: New Zealand White rabbits at dosages of 0,100, 500 or 1000 mg/kg bw/day of the test substance. The test substance was dissolved in an aqueous 3% corn starch and 0.5% Tween 80 suspension. The day of which mating was successful was designated day 0 of gestation. The animals were treated daily for 13 consecutive days from gravid day 7 through 19. Dosing volume was based on gravid day 7 body weight. The females were observed daily for clinical signs. Body weights were recorded for selected intervals during gestation. Food consumption was recorded daily. The does were sacrificed on gravid day 29; the status of the reproductive tract was determined; and the foetuses were examined for external, soft-tissue and skeletal malformations and variations.

Results showed that there were no signs of maternal toxicity, embryofoetal toxicity or teratogenicity at any dose level. The highest dose level tested (1000 mg/kg/day) was based on the EPA/OECD limit test teratology studies.

In conclusion, no maternal toxicity, embryofoetal toxicity or teratogenicity was observed at any of the dose levels tested. The NOAEL for maternal toxicity and developmental toxicity in this study was 1000 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Guideline study OECD TG 414 performed in compliance with GLP

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

All available data was assessed and the studies representing the worst-case effects was included as key study. The test substance showed no effects on developmental toxicity in rats and rabbits according to OECD TG 414 (Gilles 1989 and Meyer 1989).

Developmental toxicty in rats, Gilles 1989

The test substance was tested for embryotoxic and teratogenic action in accordance with OECD 414 and performed under GLP principles. The test substance was administered to Crl: CD® (SD) BRVAF/Plus™ rats by oral gavage as a suspension in aqueous 3% corn starch and 0.5% Tween 80 to groups of 25 mated females at dosages of 0, 100, 500 or 1000 mg/kg/day. The females were treated for 10 consecutive days from gestation day 6 through 15. The dose was based on each female’s gravid day 6 body weight. The females were monitored daily for clinical signs. Body weights and food consumption were recorded for selected intervals during gestation. The dams were sacrificed on gravid day 21; the status of the reproductive tract determined; and the foetuses examined for external, soft and tissue skeletal variations and malformations.

Results showed that treatment with the test substance caused minimal maternal toxicity at 1000 mg/kg/day indicated by a short period (gravid days 6-9) of slightly reduced body weight gain and food consumption. No embryofoetal and teratogenic toxicity was apparent at any dose level.

In conclusion, minimal maternal toxicity was evident at 1000 mg test substance/kg/day and consisted of transient reductions in body weight gained and food consumed. No embryofoetal toxicity or teratogenic effects were apparent at any of the dose levels tested. The NOAEL in this study for maternal toxicity was 500 mg/kg bw/day and for developmental toxicity was 1000 mg/kg bw/day.

Developmental toxicity in rabbits, Meyer 1989

The test substance was tested for embryotoxic and teratogenic action in accordance with OECD 414 and performed under GLP principles. The test substance was administered by oral gavage to 16 mated female Hra: New Zealand White rabbits at dosages of 0,100, 500 or 1000 mg/kg bw/day of the test substance. The test substance was dissolved in an aqueous 3% corn starch and 0.5% Tween 80 suspension. The day of which mating was successful was designated day 0 of gestation. The animals were treated daily for 13 consecutive days from gravid day 7 through 19. Dosing volume was based on gravid day 7 body weight. The females were observed daily for clinical signs. Body weights were recorded for selected intervals during gestation. Food consumption was recorded daily. The does were sacrificed on gravid day 29; the status of the reproductive tract was determined; and the foetuses were examined for external, soft-tissue and skeletal malformations and variations.

Results showed that there were no signs of maternal toxicity, embryofoetal toxicity or teratogenicity at any dose level. The highest dose level tested (1000 mg/kg/day) was based on the EPA/OECD limit test teratology studies.

In conclusion, no maternal toxicity, embryofoetal toxicity or teratogenicity was observed at any of the dose levels tested. The NOAEL for maternal toxicity and developmental toxicity in this study was 1000 mg/kg bw/day.

Justification for classification or non-classification

Based on the available data classification for toxicity to reproduction is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.