N-methyl-N-vinylacetamide

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-01-25 to 2017-01-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

28 Jul 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA, Health Effects Test Guidelines; OPPTS 870.3650: Combined Repeated Dose Toxicity Study With the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

Jul 2000

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Version / remarks:

07 September 2009

Deviations:

yes

Remarks:

applies only for the conduct of inhalation exposures, not to duration and evaluated endpoints

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-methyl-N-vinylacetamide

EC Number:

221-698-5

EC Name:

N-methyl-N-vinylacetamide

Cas Number:

3195-78-6

Molecular formula:

C5H9NO

IUPAC Name:

N-ethenyl-N-methylacetamide

Specific details on test material used for the study:

Batch identification: 68005536W0

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

The test guideline requires the rat to be used as the animal species. This rat strain was selected since extensive historical control data are available for Wistar rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males: 10-11 weeks, females: 9 weeks (ages at supply to facility day -27); males: ca. 13-15 weeks, females: 12-13 weeks (ages at experimental start day 0)
- Weight at study initiation: males: mean group values between 370.2 to 374.7 g, females: mean group values between 205.4 to 212.1 g
- Fasting period before study: no
- Housing: before randomization: 5 animals/cage (seperated by sex) in Polysulfonate cages Typ 2000P (H-Temp) supplied by TECNIPLAST, Hohenpeißenberg, Germany; after randomization: individually (in Polycarbonate cages type III supplied by TECNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany) except overnight mating (1:1, in Polycarbonate cages type III), parental animals with litters until PND 13 (in Polycarbonate cages type III) and for motor activity measurements individually in polycarbonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany with wire covers from Ehret, Emmendingen, Germany (floor area of about 800 cm2) and small amounts of bedding
- Diet: ad libitum, ground Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland
- Water: ad libitum, tap water
- Acclimation period: to facility: 27 d, to exposure procedure: 3 d
- The animals did not have access to water or feed during the exposure.

DETAILS OF FOOD AND WATER QUALITY:
The food used in the study was assayed for chemical and for microbiological contaminants. With regard to the analytical findings of chemical and microbiological contaminants and the duration of application, the diet was found to be suitable. Fed. Reg. Vol. 44, No. 91 of 09 May 1979, p. 27354 (EPA), served as a guideline for maximum tolerable chemical contaminants.The concentration of microorganisms did not exceed 1*10^5/g feed.
The drinking water is regularly assayed for chemical contaminants as well as for the presence of (pathogenic) microorganisms by the municipal authorities of Frankenthal and the Environmental Analytics Water/Steam Monitoring of BASF SE. On the basis of the analytical findings, the drinking water was found to be suitable. German Drinking Water Regulation (Trinkwasserverordnung, Bundesgesetzblatt, 05 Dec 1990) served as a guideline for maximum tolerable contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

clean air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Method of holding animals in test chamber: The animals were kept single in wire cages located in a glass-steel inhalation chamber, volume of 1.1 m³ (BASF SE).
- Source and rate of air: The central air conditioning system provided cold air of about 15 °C.
- Method of conditioning air: Cold air passed through an activated charcoal filter, was adjusted to room temperature of 20 to 24 °C and passed through a second particle filter (H13 (HEPA) Camfil Farr, Germany).
- System of generating compressed air: Compressed air was produced by an oil-free compressor (HT 6, Josef Mehrer GmbH & Co KG, Germany). For this purpose, air was filtered by an inlet air strainer and introduced into the compressor. After passing through a second ultra filter (SMF 5/3, 108 mm, Donalson), the compressed air (15 bar) was stored in a storage of 1500 or 5000 L. The compressed air was conducted to the laboratories via pipes, where the pressure was reduced to 6 bar. In the laboratory, the compressed air was taken as required.
- Temperature, humidity, pressure in air chamber: Daily relative humidities in the inhalation systems ranged between 39.2 and 60.4 %. Daily temperatures in the inhalation systems ranged between 21.7 and 24.9 °C. Test groups 0 – 3: A negative pressure was maintained by adjusting the air flow of the exhaust air system. This ensured that the laboratory was not contaminated as the result of any leakage from the inhalation chamber.
- Air flow rate: Supply air 1 (total air flow of conditioned air): 18-26 m^3/h, Supply air 2 (Compressed air): 0.8-1.2 m^3/h, Supply air 3 partial flow through generator (conditioned): 5.0-9.0 m^3/h
- Air change rate: The air change within the inhalation systems was judged to be sufficient to prevent oxygen depletion by the breathing of the animals and the concentrations of the test substance used could not have a substantial influence on oxygen partial pressure. An air change of about 20 times (test group 0, control) and 21 times (test group 1, 2 and 3) per hour can be calculated by dividing the supply air flow through the volume of each inhalation system.
- Treatment of exhaust air: The exhaust air was filtered and conducted into the exhaust air of the building.
- For same exposure conditions, the cages with the animals were rotated between the levels within each chamber.

TEST ATMOSPHERE
- Brief description of analytical method used: Please refer to "Details on analytical verification of doses and concentrations"
- Samples taken from breathing zone: no, samples were taken within the chamber between the cages

VEHICLE
- Test group 1: An aqueous solution containing about 1 % test substance was used. The solution was prepared by dissolving about 2 g test substance with 200 mL ultrapure water (w/v = 1 % solution). Homogeneity of the preparation during the generation process was ensured by stirring it in the supply container during dosing.
- Test group 2 and 3: The test substance was used unchanged.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: overnight, for max. 2 weeks
- Proof of pregnancy: sperm in vaginal smear. The day on which sperm were detected was denoted " gestation day (GD) 0" and the following day "gestation day (GD) 1"
- After 2 weeks of unsuccessful pairing no replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: with litter until PND 13 (in Polycarbonate cages type III), provided with nesting material (cellulose wadding) toward the end of gestation
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of the test substance in the atmosphere in test groups 2 and 3 were determined by gas chromatography method (GC) using online GC. Offline GC analyses were drawn twice per concentration in test groups 2 and 3 during the exposure period. Owing to the low target concentration in test group 1, the concentration in the atmosphere of this group was determined by off-line gas chromatography (GC) of absorption samples. In addition, the constancy of concentration in the test group 1 were surveyed continuously with a total hydrocarbon analyzer (FID, Testa). Background level were measured by FID in test group 0.

Duration of treatment / exposure:

6 h/d
males: 30 days
females: 56 days

Frequency of treatment:

daily
no exposure on day with functional observational battery and motor activity measurement: males day 25 (first five male animals per group), females day 45 (first five female animals per group)

Details on study schedule:

- No pups were selected for mating as a OECD 422 study was conducted.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/m³ air (nominal)

Remarks:

Test group 0

Doses / concentrations 2

Dose / conc.:

0.5 mg/m³ air (nominal)

Remarks:

Test group 1

Doses / concentrations 3

Dose / conc.:

5 mg/m³ air (nominal)

Remarks:

Test group 2

Doses / concentrations 4

Dose / conc.:

50 mg/m³ air (nominal)

Remarks:

Test group 3

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: No information provided.

Positive control:

No positive control conducted.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The clinical condition of the test animals was recorded once during the pre-exposure period and on post-exposure observation days and at least 3 times (before, during and after exposure) on exposure days. Abnormalities and changes were documented daily for each animal.
- Cage side observations checked in table No.1 were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were performed in all animals once prior to the first administration (day 0) and at weekly intervals during the administration period.
- Detailed clinical observations checked in table No.1 were included.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice. During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20. Females with litter were weighed on the day after parturition (PND 1) and on PND 4, 7, 10 and 13.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption was determined once a week for male and female parental animals. Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female F0 animals). Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0-7, 7-14, and 14-20. Food consumption of F0 females which gave birth to a litter was determined on PND 1-4, 4-7, 7-10 and 10-13.

WATER CONSUMPTION AND COMPOUND INTAKE: No

OTHER:
Furthermore haematology, clinical chemistry, neurobehavioural and immunology were anaylsed. Details for those observations are provided in IUCLID section 7.5.2.

Oestrous cyclicity (parental animals):

For all females of the pool estrous cycle normality was evaluated before the beginning of the administration period.
Estrous cycle length was evaluated by daily analysis of vaginal smear for all F0 female parental rats for a minimum of 2 weeks prior to mating. Determination was continued throughout the pairing period until the female exhibited evidence of copulation. At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female with scheduled sacrifice.

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
testis weight, epididymis weight, histology: stages of spermatogenesis in the testes and interstitial testicular cell structure

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); The surplus pups or 2 preferably female pups per litter were sacrificed under isoflurane anesthesia by decapitation.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS: yes
On PND 4 the surplus pups or 2 preferably female pups per litter were sacrificed under isoflurane anesthesia by decapitation. Blood were sampled for determination of thyroid hormone concentrations. All pups were examined externally and eviscerated; their organs were assessed macroscopically.
On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4 % formaldehyde solution.
The remaining pups were sacrificed under isoflurane by decapitation. After sacrifice, all pups were examined externally and eviscerated, and their organs were assessed macroscopically.
All stillborn pups and all pups that died before weaning were examined externally, eviscerated and their organs were assessed macroscopically.
All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.


ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: only as general observation

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: The serum levels for thyroid hormones (T4) were evaluated in some pups (details on pups see above) using Elisa.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were sacrificed on study day 30 (29 exposures) after the beginning of the administration.
- Maternal animals: All surviving animals were allowed to litter and rear their pups until day 13 after parturition.

GROSS PATHOLOGY: Yes. All parental animals were sacrificed under pentobarbitone anesthesia. The left and right brachial vessels were opened. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.

Organ weights: Were determined as listed in table No. 2 and 3.

HISTOPATHOLOGY: Yes. The organs or tissues of all parental animals were fixed in in 4 % neutral-buffered formaldehyde or in modified Davidson’s solution as listed in table No. 4. Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals were sacrificed at PND 13.
- These animals were subjected to postmortem examinations as detailed under Litter observations.

Statistics:

Statistics of the clinical examinations were performed as listed in table No. 5.
For statistics of clinical pathology means, medians and standard deviations of each test group were calculated for several parameters. In addition for blood parameters following analysis applied: For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (twosided) for the hypothesis of equal medians (* for p < 0.05, ** for p < 0.01).
For statistics of pathology means and standard deviations were calculated. In addition, for weight parameters following statistical analyses were carried out: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting pvalue was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians (* for p < 0.05, ** for p < 0.01).

Reproductive indices:

Following indices were determined:
Male mating index, Male fertility index, Female mating index, Female fertility index, Gestation index, Live birth index, Postimplantation loss

Offspring viability indices:

Following indices were determined:
Viability index, Survival index, Sex ratio, Anogenital index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

During the whole study period (pre-exposure, premating, mating, postmating as well as gestation and lactation) male and female animals of test groups 1-3 (concentration levels of 0.5, 5 or 50 mg/m^3) showed no clinical signs or findings different from the control group.
One sperm-positive female of the control group did not deliver F1 pups.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no test substance-related or spontaneous mortalities in any of the groups.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean body weights and mean body weight changes of all test-substance treated parental male and female animals (test groups 1-3) during the entire study period were comparable to the concurrent control values. The statistically significantly increased body weight change in the mid-concentration females during lactation days 4-7 was considered as spontaneous in nature and not treatment-related as no dose-response relationship occurred.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption of the high-dose F0 females (50 mg/m^3) was statistically significantly below the concurrent control values during premating days 7-14 (9 % below control). This resulted in a statistical significant decrease for the whole premating period (days 0-14, 7.7 % below control). During gestation and lactation periods, the food consumption of the high-dose F0 females was comparable to the concurrent control group. However, mean body weight of these female animals in the corresponding premating days (and also during gestation and lactation) was not affected. Therefore, the decrease of mean food consumption in the high-dose females during premating day 7-14 was considered to be in the range of normal variation and was assessed as incidental and not treatment-related.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among hematological parameters were observed.
In males of test group 3 (50 mg/m^3) platelet and absolute basophil counts were higher compared to controls. However, the values were within historical control ranges (males, platelets 639-827 Giga/L; absolute basophils 0.01-0.03 Giga/L). Therefore, these alterations were regarded as incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

No treatment-related, adverse changes among clinical chemistry parameters were observed.
In females of test group 3 (50 mg/m^3) total bile acid levels were higher compared to controls. This was the only altered clinical pathology parameters among these individuals. Therefore, it was regarded as treatment-related but not adverse (ECETOC Technical Report No. 85, 2002).
In females of test group 1 (0.5 mg/m^3) total protein and albumin levels were higher compared to controls. Both parameters were not dose-dependently changed. Therefore, this alteration was regarded as incidental and not treatment-related.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Home cage observations:
No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation.

Open field observations:
The open field observations did not reveal any test substance-related findings in male and female animals of all test groups.

Sensorimotor tests/reflexes:
There were no test substance-related findings in male and female animals of all test groups.

Quantitative Parameters:
No test substance-related impaired parameters were observed in male and female animals of all test groups.

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No statistically significant changes on motor activity data (summation of all intervals) was observed in the male and female animals of all concentration groups in comparison to the concurrent control group.

Motor activity measurement (single values) was statistically significantly above the concurrent control value in females of test group 2 (5 mg/m^3) during intervals 1 (1253.4 vs. 938.2 in control) and 11 (234.0 vs. 98.4 in control). Since the increase was not concentration-related, it was assessed as incidental and not treatment-related.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were observed in the nasal cavity with incidences and grading according to the table No. 6.
In the nasal cavity, there was degeneration/regeneration of the respiratory and transitional epithelium (level I) and of the olfactory epithelium (level II-IV). This term was used, when there was irregularity in the epithelial layer, loss of cells, loss of mucus cells (respiratory epithelium), and increased numbers of large, basophilic nuclei (regeneration). All other findings occurred either individually or were biological equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
The stages of spermatogenesis in the testes of males of the high concentration (50 mg/m^3) were comparable to those of the controls.

The female animal, which was not pregnant as well as the male mating partner did not show relevant histopathological findings.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycle data, generated during the last 2 weeks prior to mating for the F1 litter, revealed regular cycles in the females of all test groups (0, 0.5, 5 or 50 mg/m^3). The mean estrous cycle duration was similar: 4.00 days (test groups 0 and 2) and 3.97 days (test groups 1 and 3).

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

The stages of spermatogenesis in the testes of males of the high concentration (50 mg/m^3) were comparable to those of the controls.

Reproductive performance:

no effects observed

Description (incidence and severity):

Male:
For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100 % in test groups 0-3.
Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter. One control male (0 mg/m^3) did not generate pregnancy. Thus, the male fertility index ranged between 90 % and 100 % without showing any relation to dosing. This reflects the normal range of biological variation inherent in the strain of rats used for this study.

Female:
The female mating index calculated after the mating period for F1 litter was 100 % in all test groups.
The mean duration until sperm was detected (GD 0) varied between 2.3 and 3.1 days without any relation to dosing.
All female rats delivered pups or had implants in utero, except one control female did not become pregnant. The fertility index varied between 100 % in test groups 1, 2 and 3 and 90 % in test group 0. These values reflect the normal range of biological variation inherent in the strain of rats used for this study.

The mean duration of gestation was similar in all test groups (i.e. between 21.7 and 22.2 days). The gestation index was 100 % in all test groups.

Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (13.1 / 12.4 / 12.8 and 11.9 implants/dam in test groups 0-3, respectively). Furthermore, there were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any significant differences between the groups, and the mean number of F1 pups delivered per dam remained unaffected (12.6 / 12.1 / 12.2 and 11.3 pups/dam in test groups 0-3, respectively).

The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 99.1 / 99.2 / 97.5 and 99.1 % in test groups 0-3. Moreover, the number of stillborn pups was not significantly different between the test groups.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

0.5 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

50 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse systemic effects observed

3

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive performance and fertility

Effect level:

50 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

5 other: mg/m³ air (nominal)

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

There was no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

The viability index indicating pup mortality during lactation (PND 0-4) varied between 100 %/ 98.6 % /100 % and 100 % in test groups 0-3, respectively, without showing any association to treatment.
The pups surviving days indicating pup mortality during lactation (PND 4-13) was 100 % in all test groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test substance-related influence on body weights and body weight change values of F1 pups were noted in test groups 1-3 (0.5, 5 and 50 mg/m^3).
One male and one female runts were seen, each, in the control and in test group 2.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test substance-related adverse necropsy observations in any of the F1 generation pups of the different test groups.
A few F1 pups showed spontaneous findings at gross necropsy, such as situs inversus (1 male mid-dose pup) and diaphragmatic hernia (1 female control pup).

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Pup number and status at delivery:
The mean number of delivered F1 pups per dam and the rates of liveborn, stillborn, cannibalized and dead F1 pups were evenly distributed among the test groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study.

Sex ratio:
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 13 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

Anogenital distance/anogenital index:
Neither on anogenital distance nor anogenital index test substance-related effects were noted in all treated F1 offspring (test groups 1-3; 0.5, 5 and 50 mg/m^3).

Nipple/ areola anlagen:
The number of low-, mid- and high-dose male pups having areolae was not influenced by the test substance when examined on PND 13.
The percentage of all male pups having areolae was not influenced by the test substance when examined on PND 13.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No effects were observed as determined in the clinical observations.

Developmental immunotoxicity (F1)

Description (incidence and severity):

In male and female pups at PND13 (0.5, 5, 50 mg/m^3), no treatment-related alterations of T4 levels were observed.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

50 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse developmental effects observed

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Table No. 6 Histopathology P0

Nasal cavity (level I)	Male animals				Female animals
Test group (mg/m^³)	0 (0)	1 (0.5)	2 (5)	3 (50)	0 (0)	1 (0.5)	2 (5)	3 (50)
No. of animals	10	10	10	10	10	10	10	9
Degeneration/regen. transitional epithelium	0	0	1	10	0	0	0	5
Grade 1			1	10
Degeneration/regen. respiratory epithelium	0	0	3	10	0	0	2	7
Grade 1			3	7			2	5
Grade 2				3				2
Nasal cavity (level II)	Male animals				Female animals
Test group (mg/m^³)	0 (0)	1 (0.5)	2 (5)	3 (50)	0 (0)	1 (0.5)	2 (5)	3 (50)
No. of animals	10	9	10	10	10	10	10	9
Degeneration/regen. olfactory epithelium	0	0	4	8	0	0	3	9
Grade 1			4	2			3	4
Grade 2				5				2
Grade 3				1				3