cyproconazole (ISO); (2RS,3RS;2RS,3SR)-2-(4-chlorophenyl)-3-cyclopropyl-1-(1H-1,2,4-triazol-1-yl)butan-2-ol

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Feb 1986 to 1 Dec 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

1983

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

Version / remarks:

1982

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-(4-chloro-phenyl)-3-cyclopropyl-1-[1,2,4]triazol-1-yl-butan-2-ol

Cas Number:

94361-06-5

Molecular formula:

C15H18ClN3O

IUPAC Name:

2-(4-chloro-phenyl)-3-cyclopropyl-1-[1,2,4]triazol-1-yl-butan-2-ol

Test animals

Species:

rat

Strain:

Wistar

Remarks:

KFM

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: F0 8 weeks , F1: 15 weeks
- Housing: Individually, (except mating period) in MacrolonR (polycar­ bonate) cages (size 3) equipped with food and water dispensers (according to Swiss law of animal protection)
- Diet: ad libitum (renewed weekly)
- Water: Municipal water in polypropylene bottles, ad libitum renewed weekly.
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: 13 Feb 1986 to 1 Dec 1986

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Premix: 50 g of test substance were mixed with 4950 g of rat diet in a TurbulaR radial mixer for 1 hour to produce a Premix with a concentration of 1%. The Premix was prepared and renewed at monthly intervals. It was kept refrigerated.
- Final diets: Were prepared weekly by mixing Premix with additional rat diet (TurbulaR radial mixer for 1 hour), according to the following
specifications/ratio (for one 5 kg diet container).

- Diet A (4 ppm): 2 g Premix+ 4998 grams Fodder
- Diet B (20 ppm): 10 g Premix+ 4990 grams Fodder
- Diet C (120 ppm): 60 g Premix+ 4940 grams Fodder

Control animals received untreated rat diet
From May 30, 1986 and May 27, 1986 respectively the Premix and final diets were prepared in 4000 g units.

Details on mating procedure:

- M/F ratio per cage: 1 male with 1 female
- Length of cohabitation: maximum of 21 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: The female was removed on day 0 and caged individually.
- Females which failed to mate were killed 26 to 28 days after the end of the mating period and examined
- Females which failed to deliver were killed on day 25 post coitum and examined

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test substance: Samples were analysed at the start of the study and at 6 month intervals, thereafter.
Test substance in diet: Premix and triplicate aliquots of 10 grams of each experimental diet were analysed for the test substance content at the start of treatment and every second month, thereafter.

Duration of treatment / exposure:

F0: 70 days prior to mating and throughout the mating period, males additional 3 weeks after termination of mating period, females throughout gestation, parturition and lactation period.
F1: 84 days prior to mating until necropsy for all animals.

Frequency of treatment:

Continuously

Details on study schedule:

- Selection of parents from F1 generation when pups were weaned
- Age at mating of the mated animals in the study: approximately 15 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

4 ppm

Remarks:

Low dose: Equivalent to mean intake of 0.33 and 0.39 mg/kg bw/day for males and females, respectively

Doses / concentrations 2

Dose / conc.:

20 ppm

Remarks:

Mid dose: Equivalent to mean intake of 1.6 and 1.9 mg/kg bw/day for males and females, respectively

Doses / concentrations 3

Dose / conc.:

120 ppm

Remarks:

High dose: Equivalent to mean intake of 9.6 and 11.6 mg/kg bw/day for males and females, respectively

No. of animals per sex per dose:

F0: 26
F1: 26

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: based upon data from a one-generation reproduction pilot-study

Examinations

Parental animals: Observations and examinations:

CLINICAL OBSERVATIONS:
- Time schedule: All animals were examined daily for clinical signs of toxicity.

BODY WEIGHT:
- Time schedule for examinations: Individual male bodyweights were recorded at weekly intervals throughout the study. Individual female bodyweights were recorded weekly during the pre­mating period, on days 0, 7, 14 and 20 of pregnancy (p.c.) and on days 0, 7, 14 and 21 postpartum (p.p.).

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, individual food consumption was monitored at the same time as the bodyweight recording except during the mating period, in which male and females had access to the same feeder. During this period food consumption was not measured.

Oestrous cyclicity (parental animals):

During mating, vaginal smears were taken daily until sperm was found in the smear.

Sperm parameters (parental animals):

Parameters examined in F0/F1 male parental generations:
Testis and epididymis were removed and fixed in 4% formalin. Histopathological examination of all control and high dose males and any animal that failed to mate were conducted.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum
- Maximum of 4 pups/sex/litter by random selection; excess pups were killed and subjected to necropsy. Litters of less than 8 pups were not altered

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: Litter size, sex distribution, clinical signs, and malformations of pups were recorded.

Postmortem examinations (parental animals):

SACRIFICE
- The animals were killed by CO2 asphyxiation.
- Male animals: All surviving animals were killed at approx. 3 weeks after the end of the mating period
- Maternal animals: The females were killed at the time of the weaning of the F1-generation offspring (day 21 post partum)

GROSS NECROPSY
- Gross necropsy: Vagina, cervix, uterus, ovaries, testes, epididymis, seminal vesicles, prostate, coagulating gland, pituitary gland, liver and any abnormal lesions. After fixation in 4% formalin, these tissues were embedded in paraffin wax.

HISTOPATHOLOGY / ORGAN WEIGHTS
- Histopathological examination of all listed tissues of all control and high dose animals and any animal that failed to mate or failed to deliver were conducted. Additionally, the liver of all P0 low-, and mid dose males were investigated due to findings in the high-dose group.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed after weaning (day 21).
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of an examination of the major organs. Organs or tissues which showed any severe macroscopic abnormalities were removed and fixed in 4% formalin.

HISTOPATHOLOGY
Microscopic examination was undertaken if considered necessary.

Statistics:

A computer program automatically subjects all measured values to a parametric or nonparametric statistical analysis. The particular analysis chosen depends on the distribution of values.

Reproductive indices:

Copulation rate, pregnancy rate, and precoital interval were calculated.

Offspring viability indices:

In an adendum to the report, pre/perinatal loss mean incidence was calculated.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Patchy fur loss was observerd in all groups. No toxic signs or symptoms were observed in any male or female rat of the low- and mid-dose groups.
One control male showed a localised, minor skin ulceration for a period of 4 weeks during the premating period. One pregnant high dose female, which failed to deliver, showed signs of distress such as increased respiration rate and piloerection. No signs were seen in any other animal.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No parental animal died during the study. There were no unscheduled sacrifice in the control- and low-dose group except the non-pregnant ones. One female of the mid-dose group and another of the high-dose group was killed because of complete postnatal litter loss on day 1 and 5 p.p., respectively. A pregnant high-dose female which failed to deliver was sacrificed on day 25 post coitum.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

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, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Slight fatty change was noted at increased incidence in livers of high dose F0 males. No other treatment related changes were observed.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Indices reflecting mating success were comparable between treated groups and controls. Several females in all groups of both generations failed to become pregnant, however, there were no treatment related differences between treated and control groups. The fertility indices in the F0 females were comparable between treated and control groups. Numbers of implantation sites per dam did not significantly differ among groups. A slightly lower number of implantation sites was found in the high-dose F0 females, but the value remained within the historical range and is therefore considered not treatment related. The mean pregnancy length did not vary statistically significant among groups

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

20 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

Remarks on result:

other:

Remarks:

Dietary equivalent to 1.6 and 1.9 mg/kg bw/day for males and females, respectively

2

Key result

Dose descriptor:

NOAEL

Remarks:

Toxicity to reproduction

Effect level:

> 350 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Dietary equivalent to 9.6 and 11.6 mg/kg bw/day for males and females, respectively.

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Patchy fur loss was observed in all groups. No toxic signs or symptoms were observed in any male or female rat of the low- and mid-dose groups. One male of the low-dose showed blood-coloured encrustation on the lids of the left eye since the premating period. One female of the control group showed a dark left eye since the mating period. No other toxic signs or symptoms were observed in any male or female rat

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No parental animal died during the study. No female was sacrificed in the control-, low- or mid-dose group except the non-pregnant ones. One female of the high-dose group was sacrificed as a consequence of complete postnatal litter loss on day 4 p.p.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

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:

effects observed, non-treatment-related

Description (incidence and severity):

A number of common microscopic findings were observed in the organs examined. The type, incidence, and severity of these findings were considered not treatment related.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Indices reflecting mating success were comparable between treated groups and controls. Several females in all groups of both generations failed to become pregnant, however, there were no treatment related differences between treated and control groups. The fertility indices in the F1 females were comparable between treated and control groups. Numbers of implantation sites per dam did not significantly differ among groups. The mean pregnancy length did not vary statistically significant among groups.

Effect levels (P1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

(=NOEL) Systemic toxicity

Effect level:

20 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

Remarks on result:

other:

Remarks:

Dietary equivalent to 1.6 and 1.9 mg/kg bw/day for males and females, respectively

2

Key result

Dose descriptor:

NOAEL

Remarks:

Toxicity to reproduction

Effect level:

> 350 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Dietary equivalent to 9.6 and 11.6 mg/kg bw/day for males and females, respectively.

Target system / organ toxicity (P1)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

A dose-related increase in pre/perinatal mortality in the mid- and high-dose groups (13.6% and 16.3%, respectively). The pre/perinatal mortality was also considered relatively high in the control group by the author at 10.7%.
There was a corresponding slight increase in postnatal mortality (days 0 – 21 p.p) in the mid- and high-dose groups (6.6% and 8.1%). Post-natal days 0 - 4 were most affected. A further analysis of the data (reported in an addendum to the report) showed that the apparent increase in pre-perinatal and post natal mortality at the mid-dose resulted from one single F0-dam which lost 100% of its pups (12/13 pre/perinatal losses, the single surviving pup dying in the first days). The mode of calculation of the indices gives a strong weight to these losses, and since no confirmation of these effects were found in the F2 litters, they were considered not treatment-related. In the high-dose, increased post-natal losses are also due to one single dam in each generation. Nevertheless, since the loss rats for both the pre/perinatal and the post natal periods are constantly high in both generations, an effect of treatment cannot be excluded.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

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:

no effects observed

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hepatic hemorrhage was observed in 2 rats, extramedullary hemopoiesis in the liver of 1 rat, hydronephrosis in 7 rats, and renal hemorrhage in 1 rat. All these findings were considered not treatment related.

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Generation:

F1

Effect level:

20 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

viability

Remarks on result:

other:

Remarks:

Dietary equivalent to 1.6 and 1.9 mg/kg bw/day for males and females, respectively

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

effects observed, non-treatment-related

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

In the F2 litters there was a slight increase in pre/perinatal mortality in the high dose only. There was a slightly higher post-natal mortality during days 0-4 in the F2 high dose group only (7.6% greater than controls).

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

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

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Non treatment related effects were observed: Hepatic congestion in 6 rats, extramedullary hemopoiesis in the liver of 7 rats, hepatocellular fatty change in 3 rats, hydronephrosis in 40 rats, lymphoid hyperplasia in the cervical lymph node of 1 rat, and hyperemia in the cervical lymph node of 2 rats.

Other effects:

no effects observed

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Generation:

F2

Effect level:

20 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

Remarks on result:

other:

Remarks:

Dietary equivalent to 1.6 and 1.9 mg/kg bw/day for males and females, respectively

Target system / organ toxicity (F2)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Table 1. Actual substance intake in mg/kg bw/day

	4 ppm		20 ppm		120 ppm
	Males	Females	Males	Females	Males	Females
F0	0.28	0.33	1.39	1.67	8.29	9.88
F1	0.37	0.45	1.77	2.16	10.88	13.30

Table 2. Liver weights of parental animals

	males				females
	0 ppm	4 ppm	20 ppm	120 ppm	0 ppm	4 ppm	20 ppm	120 ppm
F0
body weight (g)	425	433	425	431	268	265	268	270
liver weight (g)	13.8	14.3	14.0	14.7	14.0	13.4	14.1	14.8
rel. liver weight (% of bw)	3.25	3.31	3.28	3.43*	5.26	5.05	5.25	5.49
F1
body weight (g)	470	459	464	462	273	275	269	271
liver weight (g)	16.0	15.9	16.2	16.3	14.0	14.7	14.3	14.5
rel. liver weight (% of bw)	3.42	3.48	3.50	3.52	5.12	5.35	5.31	5.36

*) P<0.05

 Table 3. Mean reproductive parameters

Parameter	Generation	0 ppm	4 ppm	20 ppm	120 ppm
Females on study	F0	26	26	26	26
	F1	26	26	26	26
Females mated (Mating index (%))	F0	26 (100)	26 (100)	26 (100)	26 (100)
	F1	26 (100)	26 (100)	26 (100)	25 (96.2)
Females pregnant (Fertility index (%))	F0	26 (100)	23 (88.5)	24 (92.3)	25 (96.2)
	F1	22 (84.6)	22 (84.6)	24 (92.3)	22 (88.0)
Females with liveborn (Gestation index (%))	F0	26 (100)	23 (100)	24 (100)	24^a)(96.0)
	F1	22 (100)	22 (100)	24 (100)	22 (100)
Mean duration of gestation (days)	F0	22.3	22.4	22.6 (increase)	22.5 (increase)
	F1	22.3	22.5	22.2	22.3
Mean implantation site per dam	F0	12.5	12.4	12.4	11.6 (decrease)
	F1	12.4	12.4	12.7	12.4

a) One female failed to deliver and was killed

Table 4. 2- generation rat litter data

Parameter	Generation	0 ppm	4 ppm	20 ppm	120 ppm
Mean live pups/dam at days					9.8(-) 7.2(-) 7.1(-) 10.9 (-) 7.2 (-) 7.1 (-)
0 4a)	F1	11.2 7.8	11.5 7.7	10.8 7.5
21		7.8	7.7	7.4
0 4a)	F2	11.0 7.7	11.7 7.7	12.0 7.8
21		7.7	7.6	7.7
Pre- and perinatal loss (%)	F1 F2	10.7 11.3	7.8 7.3	13.6(+) 5.6	16.3(+) 12.6(+)
Mean postnatal loss (%) at days 0 - 4	F1	0.3	1.6	5.6	7.6(+)
0 - 21 0 - 4 0 - 21	F2	0.3 2.2 2.2	1.6 4.2 5.9	6.6 1.4 2.9	8.1(+) 5.8(+) 7.6(+)
Sex ratio at birth (males/females)	F1 F2	0.49 0.48	0.47 0.50	0.49 0.49	0.49 0.55
Mean pup weight at birth (g)
male	F1	5.9	5.8	5.9	5.9
female		5.6	5.5	5.6	5.7
male	F2	5.8	6.0	5.8	6.0
female		5.5	5.8	5.4	5.7
Mean pup weight at day 21 p.p.
male	F1	45.8	45.4	46.4	44.2
female		44.1	44.6	44.4	43.5
male	F2	46.2	49.4	49.0	47.1
female		44.9	47.6	46.9	45.5

a) after culling
(-) Decrease

(+) Increase

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, parental toxicity for liver effects was observed at the highest dose, resulting in a NOAEL of 20 ppm. Based on a dose related increase in pre/perinatal mortality in the high-dose groups in the F0 and F1 generation, 20 ppm is considered to be the NOAEL for reproductive toxicty. This dose corresponds to 1.4 and 1.7 mg/kg bw in F0 males and females and 1.8 and 2.16 mg/kg bw/day in F1 males and females, respectively

Executive summary:

In a 2 -generation reproduction study that was performed according to OECD TG 416 and GLP principles, 26 male and 26 female KFM-Wister rats per group were exposed to the test substance in 4 groups at levels of 0, 4, 20 and 120 ppm in the diet. The test substance was administered to the parental (F0)- animals during premating (10 weeks), mating, the resulting pregnancy and through weaning their offspring. Furthermore randomly selected F1-pups (4 male + 4 female/ litter if possible) were treated in the same way as the F0 animals except the premating period which covered a period of 12 weeks. Parental (F0 and F1) body weight, food consumption, clinical signs and mortality during all stages of the study, liver weight ratios, histopathological examination of reproductive system and liver, copulation- and pregnancy rates, precoital intervals and pregnancy lengths were investigated. Weanlings (F1 and F2) litter data, pre/peri- and postnatal mortality, postnatal bodyweight development and clinical signs (days 0 - 21 postpartum), sex distribution and histopathological examination of findings were investigated.

Results showed that no treatment related clinical signs were noted in either the F-0 or F-1 parental generation and no parental animals died during treatment. The only clinical signs noted in F-0 animals were in a single high dose female that failed to deliver and showed signs of distress and increased. respiration. Clinical signs in the F-1 generation were minor and did not appear to be treatment related. There were no unscheduled deaths in the controls or low dose groups of the F0 or F1. One female of the mid-dose group and one from the F0 high dose groups were sacrificed because of total litter losses on day 1 and 5 post-partum, respectively. In addition, a single F0 high dose female, which failed to give birth, was sacrificed on day 25 post-coitum. A single high dose F1 female was sacrificed following complete postnatal litter loss on day 4 post-partum. Body weight and food consumption were comparable between treated groups and controls during the entire study period. A marginal/slight increase in relative liver weight was seen in F0 males (5.5% greater than controls) and females (4.4% greater than controls), attaining statistical significance in males only (2p > 0.05). Slight fatty change was noted at increased incidence in high dose F0 males. No other treatment related changes were observed. Indices reflecting mating success were comparable between treated groups and controls (F0+F1). Several females in all groups of both generations failed to become pregnant, however, there were no treatment related differences between treated and control groups. The fertility indices in the F0 and F1 females were comparable between treated and control groups. Numbers of implantation sites per dam did not significantly differ among groups. The mean pregnancy length in the low dose F0 and F1 females was comparable to their respective controls. An increased gestation length was observed in some mid- and high-dose F0 females. A single mid-dose animal delivered a single pup on day 24 of gestation. A single female of the high F0 group failed to deliver and was not included in the calculations of gestation length. However, this female had only a single implantation site and no foetuses at termination. In the F0 generation the females delivered on days 22 or 23, however the distribution between those two days varied among groups. Delivery on day 22 occurred for 66% of the controls and 50% of the dams in the 20 and 120 ppm groups, indicating a slight increase in the length of pregnancy in some animals of these dose groups, which may have been related to treatment. However, there was no such increase in the F1 generation dams. There was a slight reduction in mean implantation sites in the F0 high dose animals (-7%vs. controls). A decreased litter size at birth was seen in this group (-12% than controls). Neither parameter was affected in the F1 generation. Among pups in the F1 generation there was a dose-related increase in pre/perinatal mortality in the mid- and high-dose groups (13.6% and 16.3%,respectively). The per/perinatal mortality was also considered relatively high in the control group by the author at 10.7%. There was a corresponding slight increase in postnatal mortality (days 0 - 21 p.p) in the mid- and high-dose groups (6.6%c and 8.1%). Post-natal days 0 – 4 were most affected. However, the apparent increases at 20 ppm in pre/peri and post-natal mortality in the F1 litters result from one single F0-dam which lost 100% of its pups (12/13 pre/perinatal losses, the single surviving pup dying in the first 4 days). As there was no effect in the F1 generation, this event was unlikely to be treatment-related. In the F2 litters there was a slight increase in pre/perinatal mortality in the high dose only. There was a slightly higher post-natal mortality during days 0-4 in the F2 high dose group only (7.6% greater than controls). Pup weights were not affected in either F0 or F1. No treatment-related alterations were identified in pups on post-mortem examination.

In conclusion, administration of the test substance in the diet of rats in this two-generation reproduction study produced minimal signs of parental toxicity in F0 males at the highest dose tested (NOAEL 20 ppm). A slight (statistically significant) increase in relative liver weight was associated with a marginally increased incidence in liver fatty change. This observation is of questionable significance as no toxicity was seen in F0 females or F1 males and females. In addition, the 90-day rat studies indicated that the NOAEL for liver effects was in excess of 80 ppm (6.4 mg/kg bw/day). An increase in gestation in some high dose F0 females may have been related to treatment, but was not observed in the F1generation. In the F0 generation there was a slight, statistically non-significant decrease in the number of implants in the high-dose group. In addition, there was a dose related increase in pre/perinatal mortality in the high-dose groups in the F0 and F1 generation (16.3% and 12.6%, respectively). There was a corresponding slight increase in postnatal mortality (days 0 - 21 p.p) in the high-dose group of the F0 and F1 (8.1% and 7.6%, respectively. Treatment with the test substance had no effects at 4 ppm. The dose level of 20 ppm could be regarded as a NOAEL in this study if the increased gestation length and litter loss of the single 20 ppm female are disregarded. This dose corresponds to 1.4 and 1.7 mg/kg bw in F0 males and females and 1.8 and 2.16 mg/kg bw/day in F1 males and females, respectively.