1-methyl-2-pyrrolidone

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Remarks:

within a 2-generation-study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

yes

Remarks:

limited maternal information: body weight without information on body weight gain and food consumption; limited number of animals per group, protocol different from guidelines

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Source: E. I. du Pont De Nemours and Company, Richmond, Virginia, USA
- Purity: 99.9%
Impurities:
- 1.5 or isomer-dimethyl-2-pyrrolidone (986 ppm)
- N-methyl-succinimide (251 ppm)
- 2-pyrrolidinone (14 ppm)
- low boiling impurities (36 ppm), high boiling impurities (40 ppm).

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD (SD)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding North Wilmington, MA, USA
- Age at study initiation:
- first group (P0 generation): 21 days
- second group (unexposed mates for F1 generation): 64 days
- Weight at study initiation:
- first group: 32-50 g
- second group: 165-217 g
- Fasting period before study: not specified
- Housing:
- before exposure: individually, cages made of stainless steel wiremesh
- during exposure: individually, stainless steel cages placed within 750 or 900 liter glass and stainless steel chambers
- animal positions within the chambers were rotated daily
- Diet: ad libitum except during the inhalation exposures; Purina Certified Rodent Chow No. 5002 (Ralston Purina Company, St. Louis, MO, USA)
- Water: ad libitum, except during the inhalation exposures
- Acclimation period: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 (72 ± 2°F)
- Humidity (%): 40-60
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

other: nitrogen carried vapors and was mixed with dilution air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: NMP pumped through inclined tube furnace heated to 195-210°C, N2 carried vapours throught the tube
- Method of holding animals in test chamber: hold in cages
- Source and rate of air: dilution air, 700 L/min
- Temperature, humidity, oxygen concentration: 18.9 - 27.7 °C (within control animal chambers: 19.2 - 28.3°C), about 50% r.h., below 21%
- Method of oxygen monitoring: Biomarine Oxygen Analyzer (Grade 1225R)
- Treatment of exhaust air: ventilation from bottom chamber through aqueous scrubbing tower

TEST ATMOSPHERE
- Brief description of analytical method used: HP 5710A GC with nitrogen/phosphorus detector
- Samples taken from breathing zone: no

Details on mating procedure:

P0
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/2
- Length of cohabitation: 5 days
- After 5 days of unsuccessful pairing replacement of first male by another male with proven fertilit of same treatment group.
- Further matings after two unsuccessful attempts: yes,if not mated female placed with another male of same treatment group for 5 d
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal lavage referred to as day 0 of pregnancy

F1 (on day 70 PP: postpartum)
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1 (by mating with additional control animals of the opposite sex)
- Length of cohabitation: 5 days
- After 5 days of unsuccessful pairing replacement of first male by another male with proven fertilit of same treatment group.
- Further matings after two unsuccessful attempts: yes,if not mated female placed with another male of same treatment group for 5 d
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal lavage referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test atmosphere measured with HP 5710A GC with nitrogen/phosphorus detector.

Duration of treatment / exposure:

Exposure period: males: 100 d, females: 147 - 177 d
Premating exposure period (males): 85 d
Premating exposure period (females): 85 d
Duration of test: approx. 177 d

Frequency of treatment:

6 h/d, 7x/wk

Details on study schedule:

- F1 parental animals not mated until Day 77 PP after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 35 days of age.

Dose / conc.:

10.3 ppm (analytical)

Remarks:

equivalent to 41 mg/m3

Dose / conc.:

50.8 ppm (analytical)

Remarks:

equivalent to 206 mg/m3

Dose / conc.:

116.4 ppm (analytical)

Remarks:

equivalent to 478 mg/m3

No. of animals per sex per dose:

groups of 10 males and 20 females

Control animals:

yes, concurrent no treatment

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
- P0 upon arrival and twice more before exposure
- during exposure while in the chamber and during handling

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
- druing handling (remove from chamber and return into chamber)
- P0 males: weekly for recovery animals
- mated females: Day 1-21 gestation
- dams: pathological changes
- F1 rats on Days 1, 4, 14, and 21 PP: postpartum, weekly for males until euthanization; females until mating
- Mated F1: Days 1-21 GD: gestation day and 1 and 22 PP

BODY WEIGHT: Yes
- Time schedule for examinations:
- P0 upon arrival and twice more before exposure
- P0 males weekly until euthanization; females weekly until mating and on d1 and d21 of gestation
- F1 males: Days 1, 4, 14, and 22 PP, weekly until they were euthanized
- F1 females: Days 1, 4, 14, 21 PP, weekly until mating and on Day 1 and 21 GD and on Days 1 and 22 PP
- F2: Days 1 and 2 PP

Sperm parameters (parental animals):

Parameters examined in P/F1 male parental generations:
testis weight

Litter observations:

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

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
number and sex of pups, livebirths, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
no

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after breeding for the P0 generation and on Day 85 PP for the F1 generaion.
- Female animals: All surviving animals on Day 21 PP for P0 generation and on Day 2 PP for the F1 generation.

GROSS NECROPSY
- Gross changes of reproductive organs observed for F1 generation.
- Gross pathological changes observed for the pups of the P0 generation within the developmental study part (Solomon et al., 1995).

POST-MORTEM EXAMINATIONS: Yes
- Organs examined: ovarian weight

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 and 2 days of age, respectively.
- These animals were subjected to postmortem examinations as follows:

GROSS NECROPSY
- Gross necropsy consisted of reproductive organs.

Statistics:

Fisher's exact test with Cochran-Armitage (pregnancy rate, clinical signs, maternal deaths, mating, fertility, gestation indices, litter survival) 
ANOVA and Dunnett's test (gestation length, body weight data, liver weights)
Mann-Whitney U test (nidations)

Reproductive indices:

Female / Male Mating Index (%) = (Number copulating / Number cohoused) * 100
Female[a] / Male[b] Fertility Index (%) = (Number bearing[a] litters sired[b] / Number copulating) * 100
Gestation index (%) = (Number of litters with at least on life pup / Number of litters) * 100

Offspring viability indices:

Viability Index (%) = (Number of pups alive Dav 4 Pre-standardization/pooling / Dav 4 Pre-standardization/pooling) * 100
Lactation Index (%) = (Number of pups alive at Weaning [Day 21 postpartum] / Number of pups alive Day 4 after litter standardization/pooling) * 100

Clinical signs:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

- P0 rats died during the study: two in the control group and one in the 51 ppm group
- 1/2 control rats died from injuries during handling

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

Food efficiency:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

- exposure-related reduction in response to sound was noted at 116 ppm

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

No effects on reproductive ability/organs were observed.
Besides, in the devlopmental part of the study, no effects apart from slightly reduced pup body weight were observed.

Dose descriptor:

NOAEC

Effect level:

51 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Behaviour

Remarks on result:

other: equivalent to 206 mg/m3

Dose descriptor:

LOAEC

Effect level:

116 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Behaviour: exposure-related reduction in response to sound was noted

Remarks on result:

other: equivalent to 478 mg/m3

Clinical signs:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- body weight gain of the F1 offspring was reduced when both parents inhaled 116 ppm

Food efficiency:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

51 ppm

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Remarks on result:

other: equivalent to 206 mg/m3

Dose descriptor:

LOAEC

Generation:

F1

Effect level:

116 ppm

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Remarks on result:

other: equivalent to 478 mg/m3

Critical effects observed:

no

Reproductive effects observed:

no

Lowest effective dose / conc.:

51 ppm

Treatment related:

no

Table 1: Delivery and litter data of the P0 and P1 generations (ECHA, Background document to the Opinion on NMP, 2014).

 

	Dose (mg / m^3)
	0	41	206	478	478 (only females exposed)	478 (only males exposed)
PO generation
Natural delivery and litter data
Male mating index (%)	27/30 (90)	8/10 (80)	9/10 (90)	18/20 (90)	10/10 (100)	9/10 (90)
Male fertility index (%)	27/27 (100)	8/8 (100)	8/9 (88,9)	17/18 (94,4)	10/10 (100)	8/9 (88,9)
Female mating index (%)	56/58 (96,6)	18/20 (90)	18/19 (94,7)	40/40 (100)	20/20 (100)	20/20 (100)
Female fertility index (%)	53/56 (94,6)	16/18 (88,9)	15/18 (83,3)	37/40 (92,5)	15/20 (75)	17/20 (85)
Gestation index (%)	53/53 (100)	16/16 (100)	15/15 (100)	37/37 (100)	15/15 (100)	17/17 (100)
Mean gestation length N	34	14	14	19	13	15
X (days)	22,6	22,5	22,6	22,7	22,7	22,6
SE	0,08	0,14	0,14	0,11	0,13	0,12
Mean number of offspring/litter
-Born	13,7	14,0	13,7	14,2	13,9	14,7
-Born alive	13,5	13,9	13,5	14,1	13,9	14,6
-Day 4 PP preculling	13,4	13,9	13,3	13,9	13,6	14,3
-Day 4 PP postculling	8,0	7,9	7,9	8,0	8,0	7,9
-Day 14 PP	8,0	7,9	7,9	8,0	8,0	7,9
-Day 21 PP	8,0	7,8	7,9	8,0	8,0	7,9
Viability index (%)	99,3	100,0	98,6	98,3	98,0	97,7
Lactation index (%)	100,0	98,4	99,0	100,0	100,0	100,0
Sex ratio (% males)	0,53	0,46*	0,43	0,46	0,48	0,50
Offspring weight/litter (g)
-Day 1 PP	7,5	7,0*	7,1	6,7*	7,1	7,3
-Day 4 PP preculling	10,8	10,0*	10,3	9,6*	10,1	10,5
-Day 4 PP postculling	10,7	9,9*	10,2	9,6*	9,9*	10,6
-Day 14 PP	30,8	27,8*	29,5	28,7*	28,6*	32,0
-Day 21 PP	49,1	45,6*	47,4	46,9*	47,2	51,6*
F1 generation
Male mating index (%)	20/20 (100)	15/16 (93.8)	15/15 (100)	22/22 (100)
Male fertility index (%)	18/20 (90)	14/15 (93,3)	14/15 (93,3)	19/22 (86,4)
Female mating index (%)	19/20 (95)	15/16 (93,8)	15/15 (100)	22/22 (100)
Female fertility index (%)	18/19 (94,7)	14/15 (93,3)	14/15 (93,3)	19/22 (86,4)
	Dose (mg / m^3)
	0	41	206	478	478 (only females exposed)	478 (only males exposed)
Gestation index (%)	17/18 (94,4)	14/14 (100)	14/14 (100)	19/19 (100)
Data from exposed females mated to unexposed males
Mean number of offspring/litter
-Born	14,7	16,1	15,7	13,9
-Born alive	14,7	15,9	15,7	13,8
-Day 2 PP	14,6	15,9	15,6	13,8
Offspring weight/litter (g)
-Day 1 PP	6,7	6,5	6,7	6,9
-Day 2 PP	7,5	7,3	7,5	7,7
Mean number of offspring/litter
-Born	13,2	13,6	14,0	13,7
-Born alive	13,2	13,5	14,0	13,6
-Day 2 PP	13,2	13,3	14,0	13,6
Offspring weight/litter (g)
-Day 1 PP	6,4	6,3	6,4	6,5
-Day 2 PP	7,3	7,2	7,2	7,3

 * significantly different from control, p ≤ 0.05

Table 2: Final body weights, testes and ovarian weights (ECHA, Background document to the Opinion on NMP, 2014).

 

Concentration (mg / m^3)	N	Final body weight (g)	Testes weight (g)	Relative testes weight
Mean testes weight
PO generation
0	20	529 (10,5)	3,47 (0,76)	0,66 (0,016)
41	5	547 (9,8)	3,56 (0,251)	0,65 (0,041)
206	5	559 (25,4)	3,65 (0,113)	0,66 (0,018)
478	15	530 (6,7)	3,60 (0,074)	0,68 (0,017)
F1 generation
0	20	439 (11,4)	3,36 (0,094)	0,77 (0,013)
41	16	448 (5,8)	3,62 (0,139)	0,81 (0,030)
206	15	457 (11,4)	3,35 (0,082)	0,74 (0,019)
478	22	449 (7,8)	3,42 (0,049)	0,77 (0,015)
Mean ovarian weight
PO generation
0	56	369,8 (3,41)	142 (4,08)	38 (1,06)
41	16	361,8 (4,77)	131 (6,98)	36 (1,79)
206	15	360,9 (7,15)	133 (7,15)	37 (1,79)
478*	36	369,4 (4,16)	136 (6,08)	37 (1,71)
Concentration (mg / m3)	N	Final body weight (g)	Testes weight (g)	Relative testes weight
F1 generation
0	17	315,8 (4,65)	189 (6,39)	60 (2,57)
41	14	320,5 (7,40)	218 (9,79)	69 (2,75)
206	14	330,2 (8,81)	210 (8,58)	64 (2,25)
478	19	318,9 (6,27)	198 (6,55)	62 (2,33)

 

Conclusions:

The toxicological significance of the apparent reduced responsiveness of top dose animals to sound is not established, but at most it represented a slight narcotic effect, the parental NOAEC therefore appears to be 50 ppm vapor. In the offspring, there was a slight decrease in pup body weight at the highest concentration only.

Overall, the study provides no evidence of an adverse effect on any parameters of fertility.

Thus, it is assessed that the following NOAECs were achieved:

Reproductive performance and fertility:
116 ppm (LOAEC), equal to 478 mg/m3
Parental toxicity:
50 ppm, equal to 206 mg/m3

Executive summary:

In a two-generation reproduction study in rats 10 males and 20 females per dose level were exposed whole body to 0, 10 ppm (41 mg/m3), 51 ppm (206 mg/m3) or 116 ppm (478 mg/m3) of NMP vapour (Solomon et al., 1995). Sprague-Dawley rats were exposed for 6h/day, 7 days/week, for a minimum of 14 weeks. Two satellite groups, where either male or female animals were exposed, were tested additionally at 478 mg/m3.

Besides, a developmental study was conducted which is described in chapter Developmental toxicity / teratogenicity.

Animals were mated after 12 weeks of exposure to NMP vapours and both parents and offspring were examined for effects on reproductive organs /reproduction. The F1 generation was not exposed after weaning and the F2 generation was produced by mating with additional rats (non-experimental animals) of the opposite sex.

There were no effects observed for the reproducibility or the reproductive organs. For the developmental effects, apart from reduced body weight of the pups at the high dose, no adverse effects were observed (Solomon et al., 1995). This observation was in line with reduced body weight gain of the F1 generation whose parents were exposed to 478 mg/m3. The effects persisted till 21 days after birth. There is no clear dose response for this effect discussed because the effects were missing for the 206 mg/m3 dose group.

For maternal toxicity a NOAEC of 51 ppm (206 mg/m3) is proposed. However, as the only effect in maternal animals was the reduced sensitivity to sound in parental animals before mating, this effect is considered to be a systemic toxicity effect.

The developmental LOAEC is 116 ppm (478 mg/m3) as effects to the pup body weight in the developmental part of the study (Solomon et al., 1995) and reduced body weight gain for the F1 generation is described in the study.

Data source

Reference

Reference 1

Reference Type:

publication

Title:

1-Methyl-2-pyrrolidone (NMP): reproductive and developmental toxicity study by inhalation in the rat.

Author:

Solomon H.M., Burgess B.A., Kennedy Jr. G.L.,, Staples R.E.

Year:

1995

Bibliographic source:

Drug Chem. Toxicol., 18(4), 271-293

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

not specified

Deviations:

yes

Remarks:

only one concentration tested; part of a 2-generation study

Principles of method if other than guideline:

segment of a reproduction 2-generation toxicity study

GLP compliance:

yes

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: E. I. du Pont De Nemours and Company, Richmond, Virginia, USA
- Purity: 99.9%
Impurities:
- 1.5 or isomer-dimethyl-2-pyrrolidone (986 ppm)
- N-methyl-succinimide (251 ppm)
- 2-pyrrolidinone (14 ppm)
- low boiling impurities (36 ppm), high boiling impurities (40 ppm)

Specific details on test material used for the study:

- Source: E. I. du Pont De Nemours and Company, Richmond, Virginia, USA
- Purity: 99.9%
Impurities:
- 1.5 or isomer-dimethyl-2-pyrrolidone (986 ppm)
- N-methyl-succinimide (251 ppm)
- 2-pyrrolidinone (14 ppm)
- low boiling impurities (36 ppm), high boiling impurities (40 ppm)

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD (SD)BR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding North Wilmington, MA, USA
- Age at study initiation:
- first group (P0 generation): 21 days
- second group (unexposed mates for F1 generation): 64 days
- Weight at study initiation:
- first group: 32-50 g
- second group: 165-217 g
- Fasting period before study: not specified
- Housing:
- before exposure: individually, cages made of stainless steel wiremesh
- during exposure: individually, stainless steel cages placed within 750 or 900 liter glass and stainless steel chambers
- animal positions within the chambers were rotated daily
- Diet: ad libitum except during the inhalation exposures; Purina Certified Rodent Chow No. 5002 (Ralston Purina Company, St. Louis, MO, USA)
- Water: ad libitum, except during the inhalation exposures
- Acclimation period: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 (72 ± 2°F)
- Humidity (%): 40-60
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

other: nitrogen carried vapors and was mixed with dilution air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: NMP pumped through inclined tube furnace heated to 195-210°C, N2 carried vapours throught the tube
- Method of holding animals in test chamber: hold in cages
- Source and rate of air: dilution air, 700 L/min
- Temperature, humidity, oxygen concentration: 18.9 - 27.7 °C (within control animal chambers: 19.2 - 28.3°C), about 50% r.h., below 21%
- Method of oxygen monitoring: Biomarine Oxygen Analyzer (Grade 1225R)
- Treatment of exhaust air: ventilation from bottom chamber through aqueous scrubbing tower

TEST ATMOSPHERE
- Brief description of analytical method used: HP 5710A GC with nitrogen/phosphorus detector
- Samples taken from breathing zone: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test atmosphere measured with HP 5710A GC with nitrogen/phosphorus detector.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1/2
- Length of cohabitation: 5 days
- After 5 days of unsuccessful pairing replacement of first male by another male with proven fertilit of same treatment group.
- Further matings after two unsuccessful attempts: yes,if not mated female placed with another male of same treatment group for 5 d
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal lavage referred to as day 0 of pregnancy

Duration of treatment / exposure:

males: 100 days, females: 106-110 d

Frequency of treatment:

6 h/d,7d/week

Duration of test:

minimum of 14 weeks

Doses / concentrations

Dose / conc.:

116.4 ppm (analytical)

Remarks:

equal to 478 mg/m3

No. of animals per sex per dose:

10 males and 20 females per dose level

Control animals:

yes, concurrent no treatment

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
- P0 upon arrival and twice more before exposure
- during exposure while in the chamber and during handling

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
- druing handling (remove from chamber and return into chamber)
- P0 males: weekly for recovery animals
- mated females: d1-d21 gestation
- dams: pathological changes

BODY WEIGHT: Yes
- Time schedule for examinations:
- P0 upon arrival and twice more before exposure
- P0 males weekly until euthanization; females weekly until mating and on d1 and d21 of gestation

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: liver, uterus

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 resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: all per litter, except for the heads fixed in Bouin's solution
- Head and eyelid examinations: Yes: all per litter; heads fixed in Bouin's solution

Statistics:

- One way ANOVA, significant F-value resulted in Dunnett's test, nonparametric procedures resulted in Batlett's test: body weight, body weight gain, organ weight and gestational length
- Cochran-Armitage test for trend, if significant Fisher's exact followed: incidence of clinical observations
- Fisher's exact: mating, fertility, gestation indices, and for litter survival
- Jonckheere's test, two-tailed Mann-Whitney U test: Live fetuses, dead fetuses, resorptions, percent resorptions, corpora lutea, fetal weight and incidence of alterations
- test for trend and pairwise test between control and experimental groups, Cochran-Armitage and Fisher's exact test: incidence of pregnancy, clinical signs, and matemal death
- two-way ANOVA: differences among groups

SIGNIFANCE LEVEL
- alpha = 0.05 for all, except for Bartlett's test (alpha = 0.005)

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

not specified

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Three P0 rats died during the study; two in the control group and one in the 51 ppm group. One of the two control rats died from an injury received during handling.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

Food efficiency:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

Ophthalmological findings:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

An exposure-related reduction in response to sound was noted at 116 ppm, but not at either 10 or 51 ppm.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

Dead fetuses:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): - no effects compared to control animals observed

Effect levels (maternal animals)

Dose descriptor:

NOAEC

Effect level:

116 ppm

Based on:

test mat.

Basis for effect level:

behaviour (functional findings)

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

- slight but statistically significant decrease in fetal body weight was observed at 116 ppm (7% = 3.37 g versus 3.62 g in controls; p<0.025).

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

External malformations:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

Skeletal malformations:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

Visceral malformations:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

Details on embryotoxic / teratogenic effects:

No significant difference in the incidence of fetal malformations, variations or retarded development was observed in the exposed rats compared to the control.
An exposure-related, slight but statistically significant decrease in fetal body weight was observed at 116 ppm (7% = 3.37 g versus 3.62 g in controls; p<0.025).
Details on study results are given in table 1.

Effect levels (fetuses)

Dose descriptor:

LOAEC

Effect level:

116 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

yes

Lowest effective dose / conc.:

116 ppm (analytical)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

no

Relevant for humans:

not specified

Any other information on results incl. tables

Table 1: Combined results of the P0 generation of the combined 2-year-generation study and the developmental toxicity study. Groups CI and EIII for use in the developmental toxicity phase of the study.

Group	CI/II	EI	EII	EIII	EIV	EV
Exposure Level (ppm)	0	10	51	116	0	116
	0	10	51	116	116	0
Mean Number of Offspring/Litter
Born	13.7	14.0	13.7	14.2	13.9	14.7
Born Alive	13.5	13.9	13.5	14.1	13.9	14.6
Day 4 PP Preculling	13.4	13.9	13.3	13.9	13.6	14.3
Day 4 PP Postculling	8.0	7.9	7.9	8.0	8.0	7.9
Day 14 PP	8.0	7.9	7.9	8.0	8.0	7.9
Day 21 PP	8.0	7.8	7.9	8.0	8.0	7.9
Viability Index(%)	99.3	100.0	98.6	98.3	98.0	97.7
Lactation Index(%)	100.0	98.4	99.0	100.0	100.0	100.0
Sex Ratio (% Males)	0.53	0.46^a	0.43	0.46	0.48	0.50
Offspring Weight/ Litter (g)
Day 1 PP	7.5	7.0^a	7.1	6.7^a	7.1	7.3
Day 4 PP Preculling	10.8	10.0^a	l0.3	9.6^a	10.1	10.5
Day 4 PP Postculling	10.7	9.9^a	10.2	9.6^a	9.9^a	10.6
Day 14 PP	30.8	27.8^a	29.5	28.7^a	28.6^a	32.0
Day 21 PP	49.1	45.6^a	47.4	46.9^a	47.2	51.6^a
^a= Significantly different from Control Group, p ≤ 0.05 C: control group; E: exposure group

Applicant's summary and conclusion

Conclusions:

The NOAEC for maternal toxicity was 116 ppm (equivalent to 478 mg/m3) with exposure exclusively in vapor form.
Based on the reduced pup body weights the investigated concentration of 116 ppm was not a NOAEC for fetotoxicity, however, in respect to the small effect, this concentration should be considered as LOAEC for developmental toxicity.

Executive summary:

As part of a reproduction toxicity study (Solomon et al., 1995), 10 male and 20 female Sprague-Dawley rats were exposed to 0 or 116 ppm (478 mg/m³) NMP vapor starting at 34 days of age for 6h/d. For the male rats, exposure continued until day 134 of age, the end of the mating period. The females were exposed to gestation day 20 (day 140 -144). Body weights of the dams were recorded on gestation day 1 and 21, and clinical signs were monitored daily before, during and after exposure. After sacrifice, dams were examined for gross pathological changes, livers were weighed, corpora lutea were counted and resorptions were determined by staining. The fetuses were sexed and subjected to external, visceral, and skeletal examinations. The indices of reproductive performance for the NMP-exposed rats did not differ significantly from those obtained for the control rats. Rats exposed to 116 ppm (478 mg/m³) had a detectable decrease in response to sound. No other signs of NMP-related toxicity were detected among the parental rats. An exposure-related slight decrease in pup weight was detected at 116 ppm. The LOAEC for developmental toxicity was therefore 116 ppm.