1-methyl-2-pyrrolidone

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997-06-04 to 1998-06-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

May 1983

Deviations:

yes

Remarks:

Due to excessive F1 pup mortality, the high dose was reduced from 500 mg/kg bw/day to 350 mg/kg bw/day after day 126 (18 weeks of treatment). Extra observations, such as percent motility, sperm count/morphology and ophthalmoscopic examinations.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1-methyl-2-pyrrolidone

EC Number:

212-828-1

EC Name:

1-methyl-2-pyrrolidone

Cas Number:

872-50-4

Molecular formula:

C5H9NO

IUPAC Name:

1-methylpyrrolidin-2-one

Test material form:

liquid

Details on test material:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BASF AG, Ludwigshafen, Germany; tank No. 32, 26-02-97
- Analytical purity: 99.9 %
- Expiration date of the lot/batch: 27 july 1998
- Purity test date: 15 April 1997

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, under N2, in brown glass containers

FORM AS APPLIED IN THE TEST: mixed with commercial rat diets

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BASF AG, Ludwigshafen, Germany; tank No. 32, 26-02-97
- Analytical purity: 99.9 %
- Expiration date of the lot/batch: 27 july 1998
- Purity test date: 15 April 1997

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, under N2, in brown glass containers

FORM AS APPLIED IN THE TEST: mixed with commercial rat diets

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Chbb = THOM (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Karl Thomae, Biberach an der Riss, Germany
- Details on animals: females, nulliparous and non-pregnant; male and female animals were derived from different litters (written statement of the breeder)
- Age at study initiation: (P) 28 ± 1 day (at arrival) plus about 15 days (acclimation)
- Weight at study initiation: (P) Males: 196.3 g (167.0 - 222.0 g); Females: 150.5 g (130.7 - 165.7 g)
- Housing: individually in type DK III stainless steel wire mesh cages (Becker & Co., Castrop Rauxel, Germany).
Exception: from day 18 of gestation until day 14 after birth the pregnant animals and their litters were housed in Markrolon type M III cages (Becker & Co., Castrop Rauxel, Germany)
- Diet: Kliba maintenance diet rat/mouse/hamster (Klingentalmühle AG, new company name Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water: from water bottles, ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 hours / 12 hours

IN-LIFE DATES: From: 04 June 1997; To: 19 June 1998

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: test diets were prepared at intervals which guaranteed that the concentration of test substance in the diet remained stable throughout the feeding period
- Mixing appropriate amounts with: control diet

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: max. 14 days (three oestrous cycles)
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually, type III cage

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of NMP in the food over a period of up to 32 days at room temperature was proven prior to dosing.

The homogeneous distribution of the test substance in the diet was examined before the beginning of the administration period. Samples of each concentration prepared were drawn for concentration control analyses at the start of the administration period, thereafter about 1, 2, 3, 7, 10, 12, 15, 21, 22, 25, 27, 33, 39, 42, 48, 50 weeks after the beginning and at termination of the in life phase.

Duration of treatment / exposure:

Exposure period:
F0: 10 weeks premating, mating, gestation/lactation and rest period of F1a and F1b offspring
F1: after weaning during 10 weeks premating, mating, gestation/lactation and rest period F2a/F2b offspring
F2: until weaning
Premating exposure period (males): 10 weeks Premating exposure period (females): 10 weeks
Duration of test: approx. 54 weeks

Frequency of treatment:

continuous (7d/w)

Details on study schedule:

- Number of generation studies: 2
- F1 parental animals not mated until 70 days after selected from the F1 litters.
- Selection of parents from F1 generation was performed before weaning.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

160 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

After day 126 (18 weeks of treatment) the high-dose level was reduced from 500 to 350 mg/kg BW/day due to excessive mortality of the F1a pups; i.e. in the F0 parental males (from study week 17 onwards), in the F0 parental females (after weaning of the F1a pups), in the F1b pups, the F1 parents and their progeny (F2a and F2b litters) .

No. of animals per sex per dose:

25 animals

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale:
In a two-generation reproduction toxicity study (with two litters in each parental generation) conducted by EXXON Biomedical Sciences Inc. in 1991 (unpublished study), NMP was administered to Charles River CrL:CdBR (Sprague-Dawley) VAF/Plus rats in the diet at dose levels of about 0, 50, 160 and 500 mg/kg bw/day. In the Exxon study decreased pup survival and decreased reproduction and fertility (second parental generation only) were observed together with parental toxicity at the highest dose level (500 mg/kg bw/day) tested . Adverse effects on fertility/reproduction parameters observed at the lower doses were considered by the authors of this study "to be spurious findings (...) and not treatment-related". However, the US EPA concluded that the reductions in male fertility and female fecundity observed at the low- and intermediate-dose levels were biologically (although not statistically) significant and that a NOAEL was not achieved in this study.

On request of the Sponsor, dietary concentrations were selected to achieve nominal dose levels of 0, 50, 160 and 500 mg/kg bw/day for the present two-generation reproduction toxicity study in Wistar rats. As excessive pup mortality was observed in the F1a litters in this study at 500 mg/kg bw/day, it was decided to reduce the high dose level to 350 mg/kg bw/day after weaning of the surviving F1a pups for the remainder of the study; i.e. in the F0 parental males (from week 17 onwards), in the F0 parental females (after weaning of the F1a pups), in the F1b pups, the F1 parents and their progeny (F2a and F2b litters) .

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations:
In general, the body weight of the parental animals was determined on the first day of dosing and thereafter once a week at the same time of the day (in the morning). However, during gestation and lactation F0/F1 females were weighed on days 0, 7, 14 and 20 of gestation, and on days 1, 4, 7, 14 and 21 after birth. The body weight change of the animals was calculated from these results.

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
Food consumption of the F0 and F1 parents was determined regularly during premating (once weekly, in general over a period of 6 days each), and additionally during gestation and lactation periods of the F0 and F1 females.

OTHER: At least once daily a check was made for dead or moribund animals.

Oestrous cyclicity (parental animals):

Estrous cycle data were evaluated for F0 and F1 generation females over a three week period prior to mating for the first pregnancies and throughout the following mating period (up to 14 days) until evidence of mating occurred. Moreover, the estrous stage of each female was determined on the day of scheduled sacrifice.

Sperm parameters (parental animals):

Different sperm parameters (motility, sperm head count, morphology) were assessed in all F0 and F1 generation males at scheduled sacrifice or shortly thereafter.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed.
The F1a, F1b, F2a and F2b pups were sexed, and weighed on the day after birth and on days 4, 7, 14 and 21 post partum. Their viability was recorded. Sexual maturation (day of preputial separation/vaginal opening) of all pups selected to become F1 parental generation animals was determined.
Standardization of litters was not performed in litters with < 10 pups. Moreover, due to the excessive pup mortality at the high concentration (i.e. 500 mg/kg body weight/day) in the first litter (F1a) of the F0 parental rats, standardization was not performed in this test group.

Postmortem examinations (parental animals):

All F0 and F1 parental animals were assessed by gross pathology (including weight determinations of several organs) and subjected to an extensive histopathological examination, special attention being paid to the organs of the reproductive system. A quantitative assessment of primordial follicles, growing follicle and antral follicles in the ovaries was performed for all control and high dose F0 and F1 parental females. Moreover, blood was collected from all F0 and F1 generation parental animals at sacrifice and frozen for future possible analyses.

Postmortem examinations (offspring):

After scheduled sacrifice brain, spleen and thymus of 3 pups/sex and litter from the F1a, F1 b (not selected to become F1 parents), F2a and F2b pups each were weighed. Normally, the first 3 male and first 3 female pups/litter were taken for these determinations. Pup body weights routinely taken during the in-life phase on day 21 p.p. were used to calculate relative pup organ weights .
All pups were examined macroscopically at necropsy (including weight determinations of brain, spleen and thymus in 3 pups/sex/litter); certain pupswere additionally inspected for further skeletal findings. 

Statistics:

See table 1 in the section: Any other information on materials and methods incl. tables

Reproductive indices:

The following indices were calculated:
Male: male mating index, male fertility index
Female: female mating index, female fertility index, gestation index, live birth index

Offspring viability indices:

Viability index, lactation index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In all substance-treated groups a dose-related discoloration of urine in the bedding was observed.
This finding was already observed in several other toxicity studies in different species and is assessed to be a sign of systemic availability of the test substance, not an adverse effect.
No further clinical signs which might be attributed to the test substance were detected in male or female F0 generation parental animals. There were no disturbances of the general behavior noted in any of the F0 parental animals.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled mortalities in any of the F0 generation parental animals in any of the groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The high dose F0 dams showed impairments in body weight/body weight gain during gestation and lactation of F1a litters (500 mg/kg bw/day) and during the gestation period of the second litter (F1b - 350 mg/kg bw/day) with concurrent impairments in food consumption; however, the high number of complete F1a litter losses at 500 mg/kg bw/day and the impaired high dose F1a and F1b pup body weights/body weight gains may at least partly account for these impairments. Moreover, slight impairments in F1 female body weight data at 350 mg/kg bw/day during premating and F2a gestation occurred.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was impaired as discussed for the effects observed in body weights.

Food efficiency:

no effects observed

Description (incidence and severity):

The measured intakes of NMP by the different test groups (calculated on the basis of interpolated mean body weights) generally correlated well with the desired target doses.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The increased incidence of focal cystic tubular dilation noted in the F0 and F1 generation high dose males and the focal papillary calcification in F1 high dose females are indicative for a possible marginal toxic effect of NMP on the kidney.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The administration of NMP had no adverse effect on estrous cycle data of the F0 parental females.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There occurred no treatment-related effects in the different sperm parameters examined at or after the sacrifice of parental males.

Reproductive performance:

no effects observed

Description (incidence and severity):

All F0 and F1 parental rats proved to be fertile at least after one of the two mating intervals each. The observable differences between the groups concerning the different reproductive parameters are regarded to be incidental in nature and are not of toxicological or biological concern.

Details on results (P0)

See table 2 for more detailed information.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

160 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

2

Dose descriptor:

LOAEL

Effect level:

350 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

3

Dose descriptor:

NOAEL

Effect level:

350 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:

not specified

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

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

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

Description (incidence and severity):

increased mean relative kidney weights in males and females

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

increased of focal cycstic tubular dilation in the kidneys of F1 males and of focal calcification in the renal papilla of F1 females

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Effect levels (P1)

1

Dose descriptor:

NOAEL

Effect level:

160 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

gross pathology

2

Dose descriptor:

LOAEL

Effect level:

350 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

gross pathology

Target system / organ toxicity (P1)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no apparent clinical signs of toxicity in offspring.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

The administration of 500 mg NMP/kg bw/day led to a very high perinatal pup mortality rate (F1a). After decreasing the NMP dose from 500 to 350 mg/kg bw/day pup survival was still affected, but much less pronounced.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Impairments in pup body weight data occurred in the high dose progeny of the F0 and F1 parental rats (i.e. F1a, F1b, F2a and F2a litters).

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

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

There were no statistically significant differences between the substance-treated rats and the concurrent controls.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At high dose, organ weight changes secondary to the body weight impairments were noted in the progeny of F0 and F1 parental rats (i.e. F1a, F1b, F2a and F2b litters).

Gross pathological findings:

no effects observed

Description (incidence and severity):

The differences between the groups were regarded to be spontaneous in nature and not associated with the treatment of the animals.

Histopathological findings:

no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

1

Dose descriptor:

NOAEL

Generation:

F1a

Effect level:

160 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

organ weights and organ / body weight ratios

2

Dose descriptor:

LOAEL

Generation:

F1a

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

organ weights and organ / body weight ratios

3

Dose descriptor:

NOAEL

Generation:

F1b

Effect level:

160 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

organ weights and organ / body weight ratios

4

Dose descriptor:

LOAEL

Generation:

F1b

Effect level:

350 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

organ weights and organ / body weight ratios

Target system / organ toxicity (F1)

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

effects observed, treatment-related

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

350 mg/kg bw/day
- slightly increased pup mortality in both litters (viability index: F2a : 91 % vs. 96% in the control group, F2b: 89% vs. 96% in the control group; lactation index: F2a: unaffected, F2b : 94% vs. 100% in the control group)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

350 mg/kg bw/day
- lower mean body weights in both pup generations until weaning (about 7% below control on day 21 p.p.) and impaired body weight gains in these pups from day 4 p.p. up to weaning (about 6% less weight gain than in the controls)

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:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

350 mg/kg bw/day
- marginally lower absolute brain (about 2% below control) and spleen (about 7% below control) weights in both pup generations
- increased relative brain weight (about 5% above controls) in the F2a pups

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

no effects observed

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

1

Dose descriptor:

NOAEL

Generation:

F2a

Effect level:

160 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

organ weights and organ / body weight ratios

2

Dose descriptor:

LOAEL

Generation:

F2a

Effect level:

350 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

organ weights and organ / body weight ratios

3

Dose descriptor:

NOAEL

Generation:

F2b

Effect level:

160 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

organ weights and organ / body weight ratios

4

Dose descriptor:

LOAEL

Generation:

F2b

Effect level:

350 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

organ weights and organ / body weight ratios

Target system / organ toxicity (F2)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

350 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

not specified

Dose response relationship:

not specified

Any other information on results incl. tables

Table 2: Maternal detailed results.

RELEVANT PARENTAL DATA
Endpoint	Dose Group [mg/kg bw/day]
	0	50	160	500/350
Food Consumption
F0 males	-	-	-	-
F0 females	-	-	-	trans. reduced *
F1 males	-	-	-	-
F1 females	-	-	-	-
Body Weight
F0 males	-	-	-	-
F0 females	-	-	-	trans. reduced *
F1 males	-	-	-	-
F1 females	-	-	-	trans. reduced *
Body Weight Gain
F0 males	-	-	-	-
F0 females	-	-	-	trans. reduced *
F1 males	-	-	-	-
F1 females	-	-	-	trans. reduced *
Relative Kidney Weights
F0 males	0.62	0.63	0.66*	0.66*
F0 females	0.73	0.72	0.72	0.73
F1 males	0.61	0.63	0.64	0.66**
F1 females	0.75	0.75	0.77	0.78*
Histopathology
Kidney: Number and graded severity of focal cystic tubular dilation
F0 males	-	-	-	increased
F0 females	-	-	-	-
F1 males	-	-	-	increased
F1 females	-	-	-	-
Kidney: Number of focal calcification in the renal papilla
F0 males	-	-	-	-
F0 females	-	-	-	-
F1 males	-	-	-	-
F1 females	-	-	-	increased
-: no effect, trans. reduced: transiently reduced, *: p<0.05, ** p<0.01

Table 3: Indices for the maternal fertility and developmental results. 

RELEVANT REPRODUCTIVE PERFORMANCE DATA
Endpoint	Dose Group [mg/kg bw/day]
	0	50	160	500/350
Mating Index [%]
F0 males/females (F1a)	100/100	100/100	100/100	100/100
F0 males/females (F1b)	100/100	100/100	100/100	100/100
F1 males/females (F2a)	100/100	100/100	100/100	100/100
F1 males/females (F2b)	100/100	100/100	100/100	100/100
Fertility Index [%]
F0 males/females (F1a)	100/100	100/100	100/100	100/100
F0 males/females (F1b)	96/96	100/100	100/100	100/100
F1 males/females (F2a)	100/100	96/96	96/96	88/88
F1 males/females (F2b)	100/100	100/100	100/100	100/100
Gestation Index [%]
F1a litter	100	100	100	100
F1b litter	100	100	100	100
F2a litter	100	100	100	95
F2b litter	100	100	100	96
Live Birth Index [%]
F1a litter	98	97	98	92**
F1b litter	100	97	98	98
F2a litter	98	99	99	98
F2b litter	97	98	99	98
* p<0.05, ** p<0.01, Cochran-Armitage trend test or Fisher’s Exact test

 

Table 4: Results and indices of the pup developmental performance.

RELEVANT DEVELOPMENTAL DATA
Endpoint	Dose Group [mg/kg bw/day]
	0	50	160	500/350
Pups Delivered [mean]
F1a pups	13.1	13.2	13.6	12.3
F1b pups	14.0	13.6	13.1	11.0*
F2a pups	13.6	13.0	13.0	11.3
F2b pups	13.9	13.3	13.6	13.3
Viability Index [%, day 0-4 p.p.]
F1a pups	96	92*	97	18**
F1b pups	92	94	96	92
F2a pups	96	95	97	91*
F2b pups	96	95	98	89**
Lactation Index [%, day 4-21 p.p.]
F1a pups	100	98	99	88
F1b pups	100	98	100	94**
F2a pups	100	99	98	99
F2b pups	100	100	99	94**
Pups Body Weight Gain
F1a pups	-	-	-	reduced **
F1b pups	-	-	-	reduced **
F2a pups	-	-	-	reduced */**
F2b pups	-	-	-	reduced */**
Sexual Maturation
Preputial Separation	-	-	-	-
Vaginal Opening	-	-	-	-
-: no effect, *: p<0.05, ** p<0.01, p.p.: post partum

 

The stability of NMP in the food over a period of up to 32 days at room temperature was proven prior to dosing.The correctness of the homogeneous distribution in the diet and the dietary concentrations were proven. The measured intakes of NMP by the different test groups correlated generally well with the desired target doses.

 

Applicant's summary and conclusion

Conclusions:

In a two-generation reproduction toxicity studies in Wistar rats, NMP had no adverse effects on reproductive performance or fertility of the F0 or F1 parental animals of all substance-treated groups. All F0 and F1 parental rats proved to be fertile at least after one of the two mating intervals (F0 parents: F1a and F1b; F1 parents: F2a and F2b) as demonstrated by the clinical and histopathological examinations. There were signs of systemic toxicity in each of the high dose groups at 500 mg/kg bw/day and also after reduction to 350 mg/kg bw/day. Parental toxicity consisted of reduced body weight gain and food intake as well as kidney findings in form of impaired organ weight and histopathological findings. Developmental toxicity was evidenced by increased pup mortality and reduced body weight gain, including corresponding effects in the investigated organs, in pups treated at 500/350 mg/kg bw/day.

Executive summary:

In a two-generation reproduction toxicity study, groups of 25 Wistar rats per sex were given NMP via the diet at initial dose levels of 0, 50, 160 or 500 mg/kg bw/day over a 10 -week premating period and throughout the mating, gestation, lactation and a rest period between pregnancies (BASF SE, 1999). The concentrations in the diet were adjusted regularly in respect to the actual body weight gain. Due to severe pup mortality in the first litter (F1a), the highest dose level was reduced to 350 mg/kg bw/day for the further course of the study. Each generation gave birth to two litters. The parental animals for the second generation were selected from pups of the second litter (F1b).

NMP had no adverse effects on reproductive performance or fertility of the F0 or F1 parental animals of all substance-treated groups. All F0 and F1 parental rats proved to be fertile at least after one of the two mating intervals (F0 parents: F1a and F1b; F1 parents: F2a and F2b) as demonstrated by the clinical and histopathological examinations. There were signs of systemic toxicity in each of the high dose groups at 500 mg/kg bw/day and also after reduction to 350 mg/kg bw/day. Parental toxicity consisted of reduced body weight gain and food intake as well as kidney findings in form of impaired organ weight and histopathological findings. Developmental toxicity was evidenced by increased pup mortality and reduced body weight gain, including corresponding effects in the investigated organs, in pups treated at 500/350 mg/kg bw/day.

Thus, the NOAEL for reproductive performance/fertility was 350 mg/kg bw/day. The NOAEL for systemic (parental) and developmental toxicity was 160 mg/kg bw/day.

This study is acceptable and satisfies the guideline requirement for a 2-generation reproductive study (OPPTS 870.3800; OECD 416) in rats.