N-methyl-N-vinylacetamide

Endpoint summary

Description of key information

In an OECD 422 study with rats, the NOAEL for the test substance was determined to be 0.5 mg/m^3 for local toxicity after repeated application, based on degeneration/regeneration of the epithelium in the nasal cavity.

The NOAEL for the test substance was determined to be 50 mg/m^3 for systemic toxicity after repeated application, based on no adverse systemic effects up to the highest concentration.

The exposure of rats (range-finding- study) to the test substance by inhalation caused histological changes in respiratory tract, liver and kidneys.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

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

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

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

Qualifier:

according to guideline

Guideline:

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

Specific details on test material used for the study:

Batch identification: 68005536W0

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

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

clean air

Details on inhalation 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 m^3 (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.

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 draw 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)

Dose / conc.:

0 mg/m³ air (nominal)

Remarks:

Test group 0

Dose / conc.:

0.5 mg/m³ air (nominal)

Remarks:

Test group 1

Dose / conc.:

5 mg/m³ air (nominal)

Remarks:

Test group 2

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.

Observations and examinations performed and frequency:

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.

OPHTHALMOSCOPIC EXAMINATION: Yes, as part of the detailed neurological examination.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: males: at termination, females: at PND 14
- Anaesthetic used for blood collection: Yes, isoflurane
- How many animals: in the first 5 surviving parental males per group and in the first 5 females with litters (in order of delivery) per group
- Parameters checked in table No.5 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: males: at termination, females: at PND 14
- Animals fasted: Yes, least 16-20 hours
- How many animals: in the first 5 surviving parental males per group and in the first 5 females with litters (in order of delivery) per group
- Parameters checked in table No.6 were examined.

URINALYSIS: Yes, as part of detailed clinical observations.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: males day 25, females day 45
- Dose groups that were examined: all, first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group
- Battery of functions tested are checked into table No. 2-4. In addition motor activity was determined in the dark using the TSE Labmaster System (TSE Systems GmbH, Bad Homburg, Germany) with 18 infrared beams per cage. The numbers of beam interrupts were counted over 12 intervals, each lasting 5 minutes.

IMMUNOLOGY: Yes
- Time schedule for examinations: pups: on PND 13; adults: all males at termination
- How many animals: one male and one female pup per litter, all adult males
- Dose groups that were examined: all
- Parameters examined: Total Thyroxine (T4) [nmol/L] Method: Elisa

Sacrifice and pathology:

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. 8 and 9.

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. 10. Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings.

Other examinations:

Furthermore observations and examinations concerning estrous cycle, male reproduction, female reproduction and delivery and litter/pups were conducted. The details on those determinations are provided in IUCLID section 7.8.1.

Statistics:

Statistics of the clinical examinations were performed as listed in table No. 11.
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).

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.

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

Description (incidence and severity):

In parental males (test groups 1, 2 and 3; 0.5, 5, 50 mg/m^3) and in male and female pups at PND13 (test groups 11, 12 and 13; 0.5, 5, 50 mg/m^3), no treatment-related alterations of T4 levels were observed.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Absolute organ weights
All mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights
All mean relative weight parameters did not show significant differences when compared to the control group 0.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
The female animal, which was not pregnant as well as the male mating partner did not show relevant gross lesions.

Neuropathological findings:

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. 12.
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:

no effects observed

Description (incidence and severity):

Results from observations and examinations concerning estrous cycle, male reproduction, female reproduction and delivery and litter/pups are provided in IUCLID section 7.8.1.

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

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

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

5 mg/L air (nominal)

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Table No. 12 Histopathology

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

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

50 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

The guideline study is considered reliable without restriction.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

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

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

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

Qualifier:

according to guideline

Guideline:

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

Specific details on test material used for the study:

Batch identification: 68005536W0

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

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

clean air

Details on inhalation 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 m^3 (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.

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 draw 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)

Dose / conc.:

0 mg/m³ air (nominal)

Remarks:

Test group 0

Dose / conc.:

0.5 mg/m³ air (nominal)

Remarks:

Test group 1

Dose / conc.:

5 mg/m³ air (nominal)

Remarks:

Test group 2

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.

Observations and examinations performed and frequency:

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.

OPHTHALMOSCOPIC EXAMINATION: Yes, as part of the detailed neurological examination.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: males: at termination, females: at PND 14
- Anaesthetic used for blood collection: Yes, isoflurane
- How many animals: in the first 5 surviving parental males per group and in the first 5 females with litters (in order of delivery) per group
- Parameters checked in table No.5 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: males: at termination, females: at PND 14
- Animals fasted: Yes, least 16-20 hours
- How many animals: in the first 5 surviving parental males per group and in the first 5 females with litters (in order of delivery) per group
- Parameters checked in table No.6 were examined.

URINALYSIS: Yes, as part of detailed clinical observations.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: males day 25, females day 45
- Dose groups that were examined: all, first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group
- Battery of functions tested are checked into table No. 2-4. In addition motor activity was determined in the dark using the TSE Labmaster System (TSE Systems GmbH, Bad Homburg, Germany) with 18 infrared beams per cage. The numbers of beam interrupts were counted over 12 intervals, each lasting 5 minutes.

IMMUNOLOGY: Yes
- Time schedule for examinations: pups: on PND 13; adults: all males at termination
- How many animals: one male and one female pup per litter, all adult males
- Dose groups that were examined: all
- Parameters examined: Total Thyroxine (T4) [nmol/L] Method: Elisa

Sacrifice and pathology:

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. 8 and 9.

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. 10. Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings.

Other examinations:

Furthermore observations and examinations concerning estrous cycle, male reproduction, female reproduction and delivery and litter/pups were conducted. The details on those determinations are provided in IUCLID section 7.8.1.

Statistics:

Statistics of the clinical examinations were performed as listed in table No. 11.
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).

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.

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

Description (incidence and severity):

In parental males (test groups 1, 2 and 3; 0.5, 5, 50 mg/m^3) and in male and female pups at PND13 (test groups 11, 12 and 13; 0.5, 5, 50 mg/m^3), no treatment-related alterations of T4 levels were observed.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Absolute organ weights
All mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights
All mean relative weight parameters did not show significant differences when compared to the control group 0.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
The female animal, which was not pregnant as well as the male mating partner did not show relevant gross lesions.

Neuropathological findings:

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. 12.
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:

no effects observed

Description (incidence and severity):

Results from observations and examinations concerning estrous cycle, male reproduction, female reproduction and delivery and litter/pups are provided in IUCLID section 7.8.1.

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

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

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

5 mg/L air (nominal)

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Table No. 12 Histopathology

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

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

0.5 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

The guideline study is considered reliable without restriction.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a dose range finding study, groups of 5 female Wistar rats were whole-body exposed to atmospheres containing the test substance as a vapor. The target concentrations were 0, 100, 300 and 1000 mg/kg³. Effects observed were as follows:

1000 mg/mg³:

Statistically significant decrease in terminal body weight (-18%).

Statistically significant decrease of the mean absolute (-26%) weights of the adrenal glands

Statistically significant increase in absolute (+17%) and relative (+63%) weight of the kidneys

Statistically significant increase in relative (+24%) liver weight

Statistically significant increase in relative (+29%) lung weight

Statistically significant decrease in absolute (-42%) spleen weight

Macroscopically clay discoloration of all livers

Degeneration/regeneration of the transitional epithelium, respiratory epithelium, and/or olfactory epithelium (depending on level) in the nasal cavity of all animals (up to severe)

Slight to moderate atrophy of Steno’s gland in the nasal cavity (level III)

Slight hypertrophy/hyperplasia of bronchi/bronchioli (most prominent in terminal bronchioli) in the lungs of all animals

Severe dysplasia centrilobular; eosinophilic foci; moderately increased mitotic figures; minimal to slight fatty change; minimal centrilobular single cell necrosis

and slight pigment storage in the centrilobular area in the livers of all animals

Severe, diffuse hyperplasia of tubules in the kidneys of all animals

In clinical chemistry several liver parameters were changed

Increased white blood cell, neutrophils and lymphocytes in blood

300 mg/mg³:

Statistically significant decrease in terminal body weight (-10%).

Statistically significant increase in relative (+17%) weight of the kidneys

Statistically significant increase in relative (+15%) lung weight

Degeneration/regeneration of the transitional epithelium, respiratory epithelium, and/or olfactory epithelium (depending on level) in the nasal cavity of all

animals (up to moderate)

Slight hypertrophy/hyperplasia of bronchi/bronchioli (most prominent in terminal bronchioli) in the lungs of one animal

Slight to moderate dysplasia centrilobular; eosinophilic foci (one animal affected); slight to moderately increased mitotic figures; minimal fatty change; and

minimal pigment storage in the centrilobular area in the livers of all animals

Slight to moderate, diffuse hyperplasia of tubules in the kidneys of all animals

In clinical chemistry several liver parameters were changed

100 mg/mg³:

Degeneration/regeneration of the transitional epithelium (one animal), respiratory epithelium, and/or olfactory epithelium (depending on level) in the nasal

cavity of all animals (up to moderate)

Minimal dysplasia centrilobular (three animals); minimally increased mitotic figures (in one animal); minimal to slight fatty change (three animals); and minimal

pigment storage in the centrilobular area (one animal) in the livers.

The subsequent repeated dose toxicity study was conducted according to OECD 422 in rats to evaluate the toxicity profile of the test substance after inhalation exposure. Groups of 10 male and 10 female Wistar rats (F0 animals) per test group were exposed in a whole-body inhalation system to dynamic atmosphere of the test substance for 6 hours per day on each day. The duration of treatment covered a 2-week premating and 2-week mating period in both sexes, one day postmating in males, and the entire gestation and lactation period (until PND 19) of the females. The target concentrations were 0.5, 5 and 50 mg/m^3. A concurrent control group was exposed to conditioned air. Analysis of the atmospheric concentration showed that the target concentrations of 0.5, 5 and 50 mg/m^3 were met well and the atmospheres were stable during the daily exposure. All parental animals survived the duration of the study. Animals of test group 1 (0.5 mg/m^3) showed no test substance related adverse effects. Generally no test substance-related adverse effects were noted in the results of detailed clinical signs, body weights, food consumption, estrous cycle, sensory function, motor activity, urinalysis, hematology, clinical chemistry, thyroid hormone (T4, only males examined) and organ weights of animals of both sexes in the test substance-dosed groups. In histological analysis minimal degeneration/regeneration of the transitional (one male) and respiratory epithelium in the nasal cavity of level I and minimal degeneration/regeneration of the olfactory epithelium in the nasal cavity of level II to IV was observed for animals treated with 5 mg/m^3. For animals exposed to 50 mg/m^3 minimal to slight degeneration/regeneration of the transitional and respiratory epithelium in the nasal cavity of level I and minimal to moderate degeneration/regeneration of the olfactory epithelium in the nasal cavity of level II to IV was detected. Therefore the NOAEL for the test substance was determined to be 0.5 mg/m^3 for local toxicity after repeated application, based on degeneration/regeneration of the epithelium in the nasal cavity. The NOAEL for the test substance was determined to be 50 mg/m^3 for systemic toxicity after repeated application, based on no adverse systemic effects up to the highest concentration.

Justification for classification or non-classification

Based on the results of the available studies, the test item has to be classified and labelled with STOT RE cat. 1 (target organs: liver, kidney, respiratory tract) according to Regulation 1272/2008 (CLP).