1,3-bis(1-isocyanato-1-methylethyl)benzene

Endpoint summary

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Feb. 03, 2003 to Feb. 16, 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study is valid without restriction for the reproductive/developmental endpoints. However, some of the parameters measured in guideline repeated dose studies (hematologies, clinical chemistries and complete organ histopathology) were not performed. Therefore, a rating of (2) is appropriate for the repeated dose toxicity endpoints.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Manston Road, Margate, Kent
- Weight at study initiation: Males: 347-387 g; Females: 212-241 g
- Housing: 5/cage; polypropylene cages with solid floors and stainless steel tops
- Diet: Certified Rodent Diet PMI 5002; ad libitum
- Water: Mains water; ad libitum
- Acclimation period: 7 d


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2 °C
- Humidity (%): 55 ± 15 %
- Air changes (per hr): 15/h
- Photoperiod (hrs dark / hrs light): 12 h dark/12 h light

IN-LIFE DATES: From: Feb. 03, 2004 To: Mar. 19, 2004

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Preparation of the test material/vehicle formulations were performed weekly. Analysis showed test material to be stable in arachis oil for at least 14 d at ambient temperature and humidity in the dark.

VEHICLE
- Concentration in vehicle: 0, 3.75, 37.5 and 62.5 % mg/mL

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Samples of each formulation were taken on three occasions throughout the study (representing the start, middle and end of the dosing period) and analysed for achieved concentration
- Results showed that the majority of preparations to be within acceptable limits of the nominal concentrations
- Mean analytical concentrations were 129%, 102% and 110% of nominal 3.75, 37.5 and 62.5 mg/mL solutions, with the exception that the low dose formulation had a concentration above the acceptable limit on one occasion.
- The use of this formulation was not considered to influence the outcome of the study.

Duration of treatment / exposure:

19 d (males), 40-41 d (females)

Frequency of treatment:

Once daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

15 mg/kg bw/day (actual dose received)

Dose / conc.:

150 mg/kg bw/day (actual dose received)

Dose / conc.:

250 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on a range-finding study
- Rationale for animal assignment (if not random): Randomisation procedure based on stratified bw

Positive control:

Not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS (mortality and moribundity): Yes
- Time schedule: Twice daily, (once daily on weekends)


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily before and after dosing, 1 and 5 h post-dosing


BODY WEIGHT: Yes
- Time schedule for examinations: Weekly for males and females during maturation and mating period; in mated females on Day 0, 7, 14 and 20 post coitum and Days 1 and 4 post partum


FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule: Weekly for males and females during maturation period; in mated females between Days 1-7, 7-14 and 14-20 post coitum and 1-4 post partum

Sacrifice and pathology:

SACRIFICE:
- Males were euthanized after confirmation of successful mating; all surviving adults (including non-fertile animals) and offspring were euthanized on Day 5 postpartum.

EXAMINATIONS:
- All animals were examined macroscopically for internal and external abnormalities.
- Organ weights: The testes and epididymides of all adult males were weighed
- Histopathology: Following list of organs from the high dose and control adult males and females were fixed, processed and examined microscopically: coagulating glands, epididymides, prostate, seminal vesicles, testes, pituitary, ovaries, uterus/cervix and vagina

Other examinations:

REPRODUCTIVE/DEVELOPMENTAL PARAMETERS
- Parameters examined in all male parental generations: Testis weight, epididymis weight, sperm count in testes and sperm count in epididymides
- Number of corpora lutea of all ovaries from pregnant females
- Number of uterine implantation sites

Litter observations: For each litter the following was recorded:
- Number of pups born
- Number and sex of pups alive recorded daily and reported on Day 1 and 4 post partum
- Clinical condition of pups from birth to Day 4 post partum
- Individual litter weights on Day 1 and 4 post partum

Statistics:

Data were processed to give litter mean values, group mean values and standard deviations. The food conversion ratio (group mean weekly body weight gain/ food consumption) was calculated for the premating period. Adult body weight and food consumption, litter size and weight, individual pup body weight, pinna detachment, reproductive and viability indices and organ weight data were analyzed for homogeneity using Bartlett's' test, followed by a one-way analysis of variance (ANOVA). Data that were not homogeneous were subsequently analyzed using a t-test (assuming unequal variances). Dunnett's multiple comparison method was used to analyze data that were homogenous. Relative organ weights were analyzed using the Kruskal-Wallis non parametric rank sum test. Pairwise comparisons were performed using the Mann-Whitney U-test. Histopathological lesions that occurred at an overall frequency of 1 or greater were analyzed using a chi-squared test. Severity grades were analyzed using a Kruskal-Wallis one-way non-parametric ANOVA. Significant differences were reported at the p < 0.05, p < 0.01 and p < 0.001 level (if present).

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

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

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
Mortality:
- Thirteen animals were found dead or were killed in extremis during the study (four, five and four treated with 15, 150 and 250 mg/kg bw/day, respectively). All mortalities were considered to be related to mal-administration of the test material.
Clinical signs:
- At 250 mg/kg bw/d: Increased salivation pre dose and up to 1 h post dose for various durations. Isolated incidents of increased salivation 5 h post dose. Diarrhoea, diuresis, pilo-erection, hunched posture and tiptoe gait were seen sporadically.
- At 150 mg/kg bw/d: Increased salivation pre dose and up to 1 h post dose for various durations. Isolated incidents of increased salivation 5 h post dose. Pilo-erection, hunched posture, tiptoe gait were seen sporadically.
- At 15 mg/kg bw/d: Isolated incidents of increased salivation both pre and post dose. Occasional clinical signs including tiptoe gait were observed in isolated individuals during the study.


BODY WEIGHT AND WEIGHT GAIN
- At 250 mg/kg bw/d: Slight reduction in bodyweight gain in males during weeks 2, 4, 5 and 6; lower body weights in females on Day 0 of gestation and lower body weight gains throughout gestation (which resulted in lower group mean body weights on Days 7, 14 and 20 of gestation). Day 1 and 4 post partum group mean body weights of females treated with 250 mg/kg bw/day were also lower than control.
- At 150 mg/kg bw/d: The only difference observed in weights or weight gains was a slight decrease in Day 1 post partum body weight. The difference was statistically significant (p < 0.05) , but was not considered to represent a significant effect as subsequent weight gain and the Day 4 post partum body weight were not significantly different from control.
- At 15 mg/kg bw/d: There was no effect of treatment on body weight.

FOOD CONSUMPTION
- At 250 mg/kg bw/d: Food consumption of males was significantly lower than control during the first week of dosing and not affected in females during the maturation period. However, it was decreased during gestation days 1-7, 7-14 and 14-20 and lactation days 1 and 4.
- At 150 and 15 mg/kg bw/d: There was no effect of treatment on food consumption.


ORGAN WEIGHTS
- There was no effect of treatment on parental organ weight; both testes and epididymis weights (absolute and relative to body weight) of all treated groups were greater than control. The differences were significant for relative testes (p < 0.001) and epididymis weights (p < 0.05) of high dose animals. This was due to one control male with small testes (testes weight was 0.209% of body weight compared to others in the group being 0.547 - 0.763% of body weight) and epididymides (epididymis weight was 0.108% of body weight compared to others in the group being 0.205 - 0.284% of body weight) and was not considered to be treatment-related.

GROSS PATHOLOGY AND HISTOPATHOLOGY
- All animals found dead or killed in extremis (with the exception of one female at 15 mg/kg bw/d) showed macroscopic changes consistent with dosing trauma, including fluid in the thoracic cavity and fibrous adhesions in thoracic structures. At study termination, there was evidence of gas distension in four high dose males. One mid dose male also had an ulcer in the stomach.
- There were no significant gross abnormalities in treated females or in males treated with 15 mg/kg bw/d.
- No treatment-related histopathological changes were observed in the organs that were examined.

OTHER FINDINGS: Refer to 7.8.1 Reproduction/Developmental toxicity (screening), Knox et al, 2005, KL1

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: clinical signs and effects on body weight and food consumption at 250 mg/kg bw/d

Remarks on result:

other: -

Remarks:

Toxicity potentially linked to irritating properties of the substance

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

250 mg/kg bw/day (actual dose received)

System:

other: irritation type effects

Organ:

other: gastrointestinal tract

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Under the study conditions, administration of the test substance to adult male and female rats throughout maturation, mating, gestation and early lactation resulted in significant effects at 250 mg/kg bw/d. Although effects were seen at 150 mg/kg bw/d, they were not considered to be toxicologically significant and this dose was therefore considered to be the NOAEL.

Executive summary:

A repeated dose reproductive/developmental toxicity study was conducted to screen potential adverse effect of test substance on systemic toxicity and reproduction, including embryo/foetal development in the rat according to OECD Guideline 421, in compliance with GLP. The test substance was administered by gavage at concentrations of 0, 15, 150 and 250 mg/kg bw/day to groups of ten rats of either sex for a period of 19 d in males and 40 -41 d in females. On Day 5 post-partum, all surviving animals were killed and examined macroscopically for internal and external abnormalities. All adults and offspring were observed for clinical signs. Male body weights and food consumption per cage were recorded weekly throughout the study. Body weights and food consumption per cage of females were recorded weekly until mating. After mating, body weights of females were recorded on Gestation Days (GD) 0, 7, 14, and 20 and on Lactation Days (LD) 1 and 4, and food consumption of females was determined for GD 1-7, 7-14 and 14-20. Female food consumption also was recorded for the period covering LD 1-4. The litter signs and individual pup bodyweights were recorded on Days 1 and 4 post-partum. Post mortem macroscopic examinations were performed on all adults and offspring including decedents. Histopathology was carried out on reproductive organs from control and high dose group parental animals at termination. There was a total of 13 adult mortalities, distributed across all dose groups. Twelve of these were associated with dosing trauma and the nature of the test substance. The distribution of the mortalities suggested that it was not a consequence of systemic toxicity. At 250 mg/kg bw/day, there were clinical signs of reaction to a potentially irritating substance. These included increased salivation pre- and post-dosing, evidence of a reduction in bodyweight gain throughout the different phases of the study and significant reductions in food consumption. Post-mortem evaluation at termination showed limited evidence of effects on the gastro-intestinal tract. At 150 mg/kg bw/day, there were similar clinical signs of reaction to dosing and the post-mortem evaluation of adults at termination showed isolated incidents of potential gastro-intestinal effects. There were however no significant effects on bodyweight and food consumption during the in-life phase of the study. At 15 mg/kg bw/day, there were similar clinical signs of increased salivation. There were no significant findings at post-mortem macroscopic examination and no effects upon bodyweight or food consumption during the course of the study. There were no significant histopathological changes observed for the reproductive organs of adults at termination in any dose group. Under the study conditions, administration of the test substance to adult male and female rats throughout maturation, mating, gestation and early lactation resulted in significant effects at 250 mg/kg bw/day. Although effects were seen at 150 mg/kg bw/day, they were not considered to be toxicologically significant and this dose was therefore considered to be the NOAEL (Knox, 2005).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subacute

Species:

rat

System:

other: irritation type effects

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Dec. 14, 1987 to Mar. 18, 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc. (Kingston, NY)
- Age at study initiation: 68-70 d
- Housing: Individually; stainless steel, wire-mesh cages
- Diet: Pelleted feed (Pro Lab RMH 3000, Agway, Inc., Syracuse, NY) during exposure period and powdered feed during urine collection period, ad libitum
- Water: Water supplied by municipal authority, ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature: 20-22.2 °C
- Humidity (%): 32-72 %
- Photoperiod (hrs dark / hrs light): 12 h dark/12 h light

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: No data

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Stainless steel with glass windows
- Method of holding animals in test chamber: Whole body
- Temperature, humidity in air chamber: 18.5-28.9 °C, 24.9-59.1 %
- Air flow rate: 250 L/min
- Air change rate: 17 air changes/h
- Method of particle size determination: TSI Aerodynamic Particle Sizer


TEST ATMOSPHERE
- Brief description of analytical method used: At least three times a day by HPLC
- Samples taken from breathing zone: Yes


VAPOR GENERATION
- Apparatus: Syringe pump (Sage Instruments, Cambridge, MA)
- Method: Liquid test material was introduced at the top of the evaporator into a spiral groove of the inner wall and allowed to flow downward

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Reverse phase HPLC was performed using 501 Waters liquid chromatograph equipped with an SP 8400 ultraviolet/visible detector and an SP 4290 Computing Integrator.
- Detection limit: 0.014 ppm

Duration of treatment / exposure:

6 h/d, 5 d/wk for 13 wk, and for the 2 d during the 14 wk

Frequency of treatment:

Daily (6 h/d), 5 d/wk

Remarks:

Doses / Concentrations:
0.31 ± 0.061, 0.72 ± 0.096 and 1.46 ± 0.146 ppm
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
0 (air-exposed control), 0.4, 0.8 and 1.6 ppm
Basis:
other: target conc.

Remarks:

Doses / Concentrations:
0.68 ± 0.097, 1.33 ± 0.086 and 2.29 ± 0.198 ppm
Basis:
nominal conc.

No. of animals per sex per dose:

Ten

Control animals:

yes

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At the time of body weight collection and just preceding sacrifice

BODY WEIGHT: Yes
- Time schedule for examinations: Pre-exposure, once a wk during exposure and immediately preceding sacrifice

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to first exposure and following each exposure
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to terminal sacrifice
- Anaesthetic used for blood collection: Yes (Methoxyflurane)
- Animals fasted: Yes
- How many animals: All

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to terminal sacrifice
- Animals fasted: Yes
- How many animals: All

URINALYSIS: Yes
- Time schedule for collection of urine: At the end of the exposure regimen
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Organ weights: Brain, liver, kidneys, lungs and adrenal glands from all surviving animals and testis from male

HISTOPATHOLOGY: Yes

Statistics:

Bartlett's test for homogeneity of variances, ANOVA, Duncan's multiple range test or t- test

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

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:

effects observed, treatment-related

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
- Clinical signs: Reddening of the ears and paws; blepharospasm (squinting of the eyes) and alopecia (abnormal deficiency of hair) observed in 0.8 and 1.6 ppm group mice
- Mortality: 7 males and 9 females found dead in 1.6 ppm; 2 each in 0.8 ppm and 1 female in 0.4 ppm group

BODY WEIGHT AND WEIGHT GAIN
Decreased body weight gain in 0.4, 0.8 and 1.6 groups


OPHTHALMOSCOPIC EXAMINATION
- Central white opacities or diffuse cloudiness of the cornea were observed only in females in control, 0.4 and 0.8 ppm group
- Significance of the results was not clear

HAEMATOLOGY
No treatment-related effects


CLINICAL CHEMISTRY
No treatment-related effects


ORGAN WEIGHTS
Biologically significant increase in absolute lung weights in females at 0.8 ppm


GROSS PATHOLOGY
- Alopecia and color changes in the lung,
- Color change of the ears in a few of the 0.8 ppm group males and in both of the 1.6 ppm group males

HISTOPATHOLOGY: NON-NEOPLASTIC
- Necrosis, ulceration, squamous metaplasia and inflammatory changes in the nasal cavity
- Necrosis and inflammatory changes in the trachea and larynx
- Submucosal fibrosis of the trachea in one male mouse (0.8 ppm)
- Congestion and hemorrhage, necrosis, inflammatory changes, squamous metaplasia, bronchiolar submucosal fibrosis in the lungs at 1.6 and 0.8 ppm group
- Macrophage infiltration (alveolar histiocytosis) and Clara cell hypertrophy in 1 male of 0.4 ppm group
- Degeneration, necrosis, squamous metaplasia and inflammatory changes occurred in the nasal cavity of test material exposed mice sacrificed at the end of the exposure regimen
- Pulmonary fibrosis occurred in the 2 male (1.6 ppm), in 4 female (0.8 ppm) and in 1 mouse of the 0.4 ppm group

Key result

Dose descriptor:

NOAEC

Remarks on result:

not determinable

Remarks:

no NOAEC identified

Key result

Dose descriptor:

LOAEC

Effect level:

0.4 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Effects observed at all dose levels: Clinical signs; mortality; body weight; haematology; clinical chemistry; urinalysis; ophthalmoscopic examination; gross pathology; organ weights; histopathology

Key result

Critical effects observed:

not specified

Conclusions:

Under the test conditions, mice exposed to vapor of test material had evidence of toxicity at all exposure concentrations. A NOAEC could not be established, although the incidence, severity and depth of histological lesions within the respiratory tract generally decreased with decreasing exposure concentration. The LOAEC for the study can be considered to be 0.4 ppm, the lowest dose tested.

Executive summary:

A study was conducted to evaluate the toxic effects of 14 weeks of repeated exposure to vapour from test substance in mice. The study was performed according to OECD Guideline 413, in compliance with GLP. CD-1 mice(10/sex/group) received whole-body exposures for 6 h/d, 5 d/week, for 14 weeks to either filtered air or test substance vapour at target concentrations of 0 (control), 0.4, 0.8 or 1.6 ppm. All animals were monitored for toxic effects including clinical observations, body and organ weights, haematology, serum chemistry, urinalysis evaluations, ophthalmic changes, and macroscopic and microscopic evaluations. Mean analytical concentrations of 0.31, 0.72 and 1.46 ppm of test substance were obtained. Respiratory difficulty (e.g. gasping), reddening of the ears and paws, blepharospasm and alopecia were observed in mice at 0.8 and 1.6 ppm. The incidence of mortality was 5, 20, and 80% for the 0.4, 0.8, and 1.6 ppm groups, respectively. Effects on body weight gain were generally concentration related, being depressed at 1.6 ppm and sporadically depressed at 0.8 and 0.4 ppm. No concentration-related changes in haematology, serum chemistry and urinalysis parameters were observed. At necropsy, the principal changes included pulmonary congestion and alopecia. Biologically- significant organ weight changes in the lungs, absolute and/or relative lung weight values were increased for female mice at 0.8 ppm. In the nasal cavity, the lesions included necrosis, ulceration, squamous metaplasia and inflammatory changes; necrosis and inflammatory changes generally occurred in the larynx and trachea; pulmonary changes included congestion, haemorrhage, necrosis, inflammation and in several of the animals, bronchiolar submucosal fibrosis. Inflammatory changes and fibrosis in the lungs were seen at all dose levels. Under the test conditions, mice exposed to vapor of test substance had evidence of toxicity at all exposure concentrations. A NOEC could not be established, although the incidence, severity and depth of histological lesions within the respiratory tract generally decreased with decreasing exposure concentration. The LOAEC for the study can be considered to be 0.4 ppm, the lowest dose tested (Klonne, 1990).

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Dec. 14, 1987 to Mar. 18, 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc. (Kingston, NY)
- Age at study initiation: 69 d
- Housing: Individually; stainless steel, wire-mesh cages
- Diet: Pelleted feed (Pro Lab RMH 3000, Agway, Inc., Syracuse, NY) during exposure period and powdered feed during urine collection period, ad libitum
- Water: Water supplied by municipal authority, ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature: 20-22.2 °C
- Humidity (%): 32-72 %
- Photoperiod (hrs dark / hrs light): 12 h dark/12 h light

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: No data

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Stainless steel with glass windows
- Method of holding animals in test chamber: Whole body
- Temperature, humidity in air chamber: 18.5-28.9 °C, 24.9-59.1 %
- Air flow rate: 250 L/min
- Air change rate: 17 air changes/h
- Method of particle size determination: TSI Aerodynamic Particle Sizer


TEST ATMOSPHERE
- Brief description of analytical method used: At least three times a day by HPLC.
- Samples taken from breathing zone: Yes


VAPOR GENERATION
- Apparatus: Syringe pump (Sage Instruments, Cambridge, MA)
- Method: Liquid test material was introduced at the top of the evaporator into a spiral groove of the inner wall and allowed to flow downward

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Reverse phase HPLC was performed using 501 Waters liquid chromatograph equipped with an SP 8400 ultraviolet/visible detector and an SP 4290 Computing Integrator.
- Detection limit: 0.014 ppm

Duration of treatment / exposure:

6 h/d, 5 d/wk for 13 wk, and for the 2 d during the 14 wk

Frequency of treatment:

Daily (6 h/d), 5 d/wk

Remarks:

Doses / Concentrations:
0, 0.31 ± 0.061, 0.72 ± 0.096 and 1.46 ± 0.146 ppm
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
0 (control), 0.4, 0.8 and 1.6 ppm
Basis:
other: target conc.

No. of animals per sex per dose:

Ten

Control animals:

yes

Details on study design:

- Rationale for animal assignment: Animals were assigned by computer-based randomization program

Positive control:

No data

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At the time of body weight collection and just preceding sacrifice


BODY WEIGHT: Yes
- Time schedule for examinations: Pre-exposure, once a wk during exposure and immediately preceding sacrifice


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to first exposure and following each exposure
- Dose groups that were examined: All


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to terminal sacrifice
- Anaesthetic used for blood collection: Yes (Methoxyflurane)
- Animals fasted: Yes
- How many animals: All


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to terminal sacrifice
- Animals fasted: Yes
- How many animals: All


URINALYSIS: Yes
- Time schedule for collection of urine: At the end of the exposure regimen
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see Appendix 4)
- Organ weights: Brain, liver, kidneys, lungs and adrenal glands from all surviving animals and testis from male

HISTOPATHOLOGY: Yes (see Appendix 4) (including epididymes, prostate, testes, uterus and ovaries)

Statistics:

Bartlett's test for homogeneity of variances, ANOVA, Duncan's multiple range test or t- test

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

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:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
- Clinical signs: Respiratory difficulties (gasping, audible respiration), reddening of the ears and paws
- Mortality: 15 % in 1.6 ppm group of rats (males only)

BODY WEIGHT AND WEIGHT GAIN
Decreased body weight gain in 0.4, 0.8 and 1.6 ppm groups

OPHTHALMOSCOPIC EXAMINATION
No changes

HAEMATOLOGY
Increased mean corpuscular volume (males) and erythrocyte count (females) in 0.4, 0.8 and 1.6 ppm group rats

CLINICAL CHEMISTRY
Decreased albumin (0.8 and 0.4 ppm) and glucose concentration (0.4 ppm) in females

URINALYSIS
Decreases in the urine volume for the 0.8 and 1.6 ppm group females

ORGAN WEIGHTS
Increased absolute and relative lung weights in 1.6 ppm group

GROSS PATHOLOGY
Color change and the presence of emphysematous lungs in the 1.6 ppm group

HISTOPATHOLOGY: NON-NEOPLASTIC
- Necrosis, ulceration, squamous metaplasia, and inflammatory changes occurred in the nasal cavity
- Necrosis and inflammatory changes in the trachea and larynx
- Hemorrhage, edema, necrosis, submucosal fibrosis and fibrous polyps, which projected into the lumen, in the lung
- Squamous metaplasia and inflammatory changes throughout the respiratory tract, with bronchiolar submucosal fibrosis in the lungs (at 1.6 ppm)
- Squamous metaplasia and inflammatory infiltrates in upper respiratory tract (at 0.4 and 1.6 ppm), bronchiolar submucosal fibrosis (at 0.8 ppm)

Key result

Dose descriptor:

NOAEC

Remarks on result:

not determinable

Remarks:

no NOAEC identified

Key result

Dose descriptor:

LOAEC

Effect level:

0.4 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Effects observed at all dose levels: Clinical signs; mortality; body weight; haematology; clinical chemistry; urinalysis; gross pathology; organ weights; histopathology

Key result

Critical effects observed:

not specified

Conclusions:

Under the test conditions, rats exposed to vapor of the test material had evidence of toxicity at all exposure concentrations. A NOAEC could not be established, although the incidence, severity and depth of histological lesions within the respiratory tract generally decreased with decreasing exposure concentration. The LOAEC for the study can be considered to be 0.4 ppm, the lowest dose tested.

Executive summary:

A study was conducted to evaluate the effects of 14 weeks of repeated exposure to vapour of the test substance in rats according to a method equivalent or similar to OECD Guideline 413, in compliance with GLP. Sprague-Dawley rats (10/sex/group) received whole-body exposures for 6 h/d, 5 d/week, for 13 week to either filtered air or test substance vapour at target concentrations of 0 (control), 0.4, 0.8 or 1.6 ppm. All animals were monitored for toxic effects including clinical observations, body and organ weights, haematology, serum chemistry, urinalysis evaluations, ophthalmic examinations, and macroscopic and microscopic evaluations. Mean analytical concentrations of 0.31, 0.72 and 1.46 ppm of test substance were obtained. The incidence of mortality at 1.6 ppm was 15%. Respiratory difficulty and reddening of ears and paws were observed at 0.8 and 1.6 ppm. Effects on body weight gain were generally concentration-related, being depressed at 1.6 ppm and sporadically depressed at 0.8 and 0.4 ppm. Many of the haematology and serum chemistry parameters were abnormal at 1.6 ppm. Increased mean corpuscular volume and erythrocyte count, and decreased albumin concentration, glucose concentration and urine volume were observed at 0.4 or 0.8 ppm. At necropsy, the principal observations included a colour change (congestion) and emphysematous lungs at 1.6 ppm. Absolute and/or relative lung weight values were increased for rats at 1.6 ppm. In the nasal cavity, lesions included necrosis, ulceration, squamous metaplasia and inflammatory changes; necrosis and inflammatory changes generally occurred in the larynx and trachea; pulmonary changes included congestion, haemorrhage, necrosis, inflammation and in several of the animals, bronchiolar submucosal fibrosis. At 1.6 ppm, squamous metaplasia and inflammatory changes were observed throughout the respiratory tract, with bronchiolar submucosal fibrosis. At 0.4 and 0.8 ppm, exposure-related lesions were principally found in upper respiratory tract and involved squamous metaplasia and inflammation. Under the test conditions, rats exposed to vapour of the test substance had evidence of toxicity at all exposure concentrations. A NOAEC was not established, although the incidence, severity and depth of histological lesions within the respiratory tract generally decreased with decreasing exposure concentration. The LOAEC for the study can be considered to be 0.4 ppm, the lowest dose tested (Klonne, 1990).

Reference 3

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Charles River Breeding Laboratories, New York 12484
- Age at study initiation: Male: 8 wk, female: 9 wk
- Weight at study initiation: Male: 304-336 g; Female: 199-235 g
- Housing: Individually housed in stainless steel wire mesh cages
- Diet (e.g. ad libitum): Purina Certified Rodent Chow #5002, ad libitum
- Water (e.g. ad libitum): Automated watering system, ad libitum
- Acclimation period: 5 wk

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-70 %
- Photoperiod (hrs dark / hrs light): 12h dark/12 h light

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

not specified

Vehicle:

other: acetone

Remarks on MMAD:

MMAD / GSD: MMAD: 0.9-1.5 microns
GSD: 1.5-1.9

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Glass and stainless steel exposure chambers
- Source and rate of air: Matheson pressure gauze, Nupro metering valve and Dwyer flowmeter
- System of generating particulates/aerosols: Atomizing nozzle (Spraying Systems Co.; 1/4" JSS nozzle with a #1A spray set-up) with glass syringe mounted in a Sage Syringe Pump (Model #352 or #341)
- Method of particle size determination: TSI Aerodynamic Particle sizer equipped with Diluter
- Air flow rate: 200-212 L/min
- Air change rate: 4.7-5.0 min


TEST ATMOSPHERE
- Brief description of analytical method used: Samples collected using impingers and analysed by HPLC
- Samples taken from breathing zone: Yes


VEHICLE
- Composition of vehicle: Acetone
- Lot/batch no. of vehicle: 856554

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Analysis of test material in impingers by liquid chromatography
- Limit of detection: 2.78 ng injected (50 µL of a 0.0555 µg/mL standard)

Duration of treatment / exposure:

6 h/d, 5d/wk for 4 wk

Frequency of treatment:

Daily (6 h/d), 5 d/wk

Remarks:

Doses / Concentrations:
0, 0.38, 1.5 and 4.4 mg/m3
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
0, 0.5, 1.5 and 5.0 mg/m3
Basis:
other: Target conc.

No. of animals per sex per dose:

Five

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment: Ramdomly assigned by computerized randomization

Positive control:

Not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly


BODY WEIGHT: Yes
- Time schedule for examinations: Once pretest and on Day 1, 5, 8, 12, 15, 19, 22, 26 and 29


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 29
- How many animals: 5/sex/group
- Parameters examined: Hemoglobin concentration, hematocrit, erythrocyte count, clotting time, total and differential leukocyte counts

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: No
- How many animals: 5/sex/group
- Parameters examined: Serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT); blood urea, nitrogen, creatinine, fasting glucose, total protein, sodium, potassium, chloride, calcium and inorganic phosphorus

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes (including gonads, ovaries and testes with epididymes)

Statistics:

Anova, Bartletts, Dunnets, Kruskal- Wallis, Regression analysis, Jonckheere's statistic and Summed Rank Test

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
- Clinical signs: Decrease in the activity at all dose levels, increased salivation at 1.5 and 5 mg/m3 and closed eyes at 5 mg/m3
- Mortality: None


BODY WEIGHT AND WEIGHT GAIN
No significant changes in body weight except decreased body weight gain in males at 5.0 mg/m3


HAEMATOLOGY
No treatment-related effects


CLINICAL CHEMISTRY
Significant elevation in serum calcium and phosphorous in males at 5 mg/m3


ORGAN WEIGHT
Increased relative lung weight in females at 5 mg/m3


GROSS PATHOLOGY/ HISTOPATHOLOGY (NON-NEOPLASTIC)
- Evidence of discoloured lungs (red foci) at 0.5 (in 1 male), 1.5 (in 1 male and 1 female) and 5 mg/m3 (in 4 males)
- Microscopically, the significant findings were subacute/chronic inflammation in the lungs and appearance of hyperplastic and metaplastic changes in the bronchi at 5 mg/m3

Key result

Dose descriptor:

NOAEC

Effect level:

1.5 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Key result

Critical effects observed:

not specified

Conclusions:

Under the test conditions, the NOAEC of the test material was considered to be 1.5 mg/m3 in rat.

Executive summary:

A study was conducted to evaluate the inhalation toxicity of the test substance in rat according to a method equivalent or similar to OECD Guideline 412, in compliance with GLP. The test substance as a respirable aerosol was administered by inhalation to Sprague-Dawley rats (5/sex/group) for 6 h/d, 5 d/week, for 4 weeks at target concentrations of 0, 0.5, 1.5 and 5.0 mg/m3 (measured concentrations of 0 (acetone vapour), 0.38, 1.5 and 4.4 mg/m3). Animals were given detailed physical examinations. Body weight, organ weights, haematology and clinical chemistry were observed and histopathological examinations were conducted. Increased salivation, closed eyes, decreased body weight gain and elevated serum calcium/phosphorus levels (in males), increased relative lung weight (in females) and pathological changes in lungs (subacute/chronic inflammation in the lungs and appearance of hyperplastic and metaplastic changes in the bronchi) were observed at 5.0 mg/m3. Under the study conditions, the NOAEC of the test substance was considered to be 1.5 mg/m3 in rat (Rinehart, 1987).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Dec. 14, 1987 to Mar. 18, 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc. (Kingston, NY)
- Age at study initiation: 68-70 d
- Housing: Individually; stainless steel, wire-mesh cages
- Diet: Pelleted feed (Pro Lab RMH 3000, Agway, Inc., Syracuse, NY) during exposure period and powdered feed during urine collection period, ad libitum
- Water: Water supplied by municipal authority, ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature: 20-22.2 °C
- Humidity (%): 32-72 %
- Photoperiod (hrs dark / hrs light): 12 h dark/12 h light

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: No data

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Stainless steel with glass windows
- Method of holding animals in test chamber: Whole body
- Temperature, humidity in air chamber: 18.5-28.9 °C, 24.9-59.1 %
- Air flow rate: 250 L/min
- Air change rate: 17 air changes/h
- Method of particle size determination: TSI Aerodynamic Particle Sizer


TEST ATMOSPHERE
- Brief description of analytical method used: At least three times a day by HPLC
- Samples taken from breathing zone: Yes


VAPOR GENERATION
- Apparatus: Syringe pump (Sage Instruments, Cambridge, MA)
- Method: Liquid test material was introduced at the top of the evaporator into a spiral groove of the inner wall and allowed to flow downward

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Reverse phase HPLC was performed using 501 Waters liquid chromatograph equipped with an SP 8400 ultraviolet/visible detector and an SP 4290 Computing Integrator.
- Detection limit: 0.014 ppm

Duration of treatment / exposure:

6 h/d, 5 d/wk for 13 wk, and for the 2 d during the 14 wk

Frequency of treatment:

Daily (6 h/d), 5 d/wk

Remarks:

Doses / Concentrations:
0.31 ± 0.061, 0.72 ± 0.096 and 1.46 ± 0.146 ppm
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
0 (air-exposed control), 0.4, 0.8 and 1.6 ppm
Basis:
other: target conc.

Remarks:

Doses / Concentrations:
0.68 ± 0.097, 1.33 ± 0.086 and 2.29 ± 0.198 ppm
Basis:
nominal conc.

No. of animals per sex per dose:

Ten

Control animals:

yes

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At the time of body weight collection and just preceding sacrifice

BODY WEIGHT: Yes
- Time schedule for examinations: Pre-exposure, once a wk during exposure and immediately preceding sacrifice

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to first exposure and following each exposure
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to terminal sacrifice
- Anaesthetic used for blood collection: Yes (Methoxyflurane)
- Animals fasted: Yes
- How many animals: All

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to terminal sacrifice
- Animals fasted: Yes
- How many animals: All

URINALYSIS: Yes
- Time schedule for collection of urine: At the end of the exposure regimen
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Organ weights: Brain, liver, kidneys, lungs and adrenal glands from all surviving animals and testis from male

HISTOPATHOLOGY: Yes

Statistics:

Bartlett's test for homogeneity of variances, ANOVA, Duncan's multiple range test or t- test

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

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:

effects observed, treatment-related

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
- Clinical signs: Reddening of the ears and paws; blepharospasm (squinting of the eyes) and alopecia (abnormal deficiency of hair) observed in 0.8 and 1.6 ppm group mice
- Mortality: 7 males and 9 females found dead in 1.6 ppm; 2 each in 0.8 ppm and 1 female in 0.4 ppm group

BODY WEIGHT AND WEIGHT GAIN
Decreased body weight gain in 0.4, 0.8 and 1.6 groups


OPHTHALMOSCOPIC EXAMINATION
- Central white opacities or diffuse cloudiness of the cornea were observed only in females in control, 0.4 and 0.8 ppm group
- Significance of the results was not clear

HAEMATOLOGY
No treatment-related effects


CLINICAL CHEMISTRY
No treatment-related effects


ORGAN WEIGHTS
Biologically significant increase in absolute lung weights in females at 0.8 ppm


GROSS PATHOLOGY
- Alopecia and color changes in the lung,
- Color change of the ears in a few of the 0.8 ppm group males and in both of the 1.6 ppm group males

HISTOPATHOLOGY: NON-NEOPLASTIC
- Necrosis, ulceration, squamous metaplasia and inflammatory changes in the nasal cavity
- Necrosis and inflammatory changes in the trachea and larynx
- Submucosal fibrosis of the trachea in one male mouse (0.8 ppm)
- Congestion and hemorrhage, necrosis, inflammatory changes, squamous metaplasia, bronchiolar submucosal fibrosis in the lungs at 1.6 and 0.8 ppm group
- Macrophage infiltration (alveolar histiocytosis) and Clara cell hypertrophy in 1 male of 0.4 ppm group
- Degeneration, necrosis, squamous metaplasia and inflammatory changes occurred in the nasal cavity of test material exposed mice sacrificed at the end of the exposure regimen
- Pulmonary fibrosis occurred in the 2 male (1.6 ppm), in 4 female (0.8 ppm) and in 1 mouse of the 0.4 ppm group

Key result

Dose descriptor:

NOAEC

Remarks on result:

not determinable

Remarks:

no NOAEC identified

Key result

Dose descriptor:

LOAEC

Effect level:

0.4 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Effects observed at all dose levels: Clinical signs; mortality; body weight; haematology; clinical chemistry; urinalysis; ophthalmoscopic examination; gross pathology; organ weights; histopathology

Key result

Critical effects observed:

not specified

Conclusions:

Under the test conditions, mice exposed to vapor of test material had evidence of toxicity at all exposure concentrations. A NOAEC could not be established, although the incidence, severity and depth of histological lesions within the respiratory tract generally decreased with decreasing exposure concentration. The LOAEC for the study can be considered to be 0.4 ppm, the lowest dose tested.

Executive summary:

A study was conducted to evaluate the toxic effects of 14 weeks of repeated exposure to vapour from test substance in mice. The study was performed according to OECD Guideline 413, in compliance with GLP. CD-1 mice(10/sex/group) received whole-body exposures for 6 h/d, 5 d/week, for 14 weeks to either filtered air or test substance vapour at target concentrations of 0 (control), 0.4, 0.8 or 1.6 ppm. All animals were monitored for toxic effects including clinical observations, body and organ weights, haematology, serum chemistry, urinalysis evaluations, ophthalmic changes, and macroscopic and microscopic evaluations. Mean analytical concentrations of 0.31, 0.72 and 1.46 ppm of test substance were obtained. Respiratory difficulty (e.g. gasping), reddening of the ears and paws, blepharospasm and alopecia were observed in mice at 0.8 and 1.6 ppm. The incidence of mortality was 5, 20, and 80% for the 0.4, 0.8, and 1.6 ppm groups, respectively. Effects on body weight gain were generally concentration related, being depressed at 1.6 ppm and sporadically depressed at 0.8 and 0.4 ppm. No concentration-related changes in haematology, serum chemistry and urinalysis parameters were observed. At necropsy, the principal changes included pulmonary congestion and alopecia. Biologically- significant organ weight changes in the lungs, absolute and/or relative lung weight values were increased for female mice at 0.8 ppm. In the nasal cavity, the lesions included necrosis, ulceration, squamous metaplasia and inflammatory changes; necrosis and inflammatory changes generally occurred in the larynx and trachea; pulmonary changes included congestion, haemorrhage, necrosis, inflammation and in several of the animals, bronchiolar submucosal fibrosis. Inflammatory changes and fibrosis in the lungs were seen at all dose levels. Under the test conditions, mice exposed to vapor of test substance had evidence of toxicity at all exposure concentrations. A NOEC could not be established, although the incidence, severity and depth of histological lesions within the respiratory tract generally decreased with decreasing exposure concentration. The LOAEC for the study can be considered to be 0.4 ppm, the lowest dose tested (Klonne, 1990).

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Dec. 14, 1987 to Mar. 18, 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc. (Kingston, NY)
- Age at study initiation: 69 d
- Housing: Individually; stainless steel, wire-mesh cages
- Diet: Pelleted feed (Pro Lab RMH 3000, Agway, Inc., Syracuse, NY) during exposure period and powdered feed during urine collection period, ad libitum
- Water: Water supplied by municipal authority, ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature: 20-22.2 °C
- Humidity (%): 32-72 %
- Photoperiod (hrs dark / hrs light): 12 h dark/12 h light

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: No data

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Stainless steel with glass windows
- Method of holding animals in test chamber: Whole body
- Temperature, humidity in air chamber: 18.5-28.9 °C, 24.9-59.1 %
- Air flow rate: 250 L/min
- Air change rate: 17 air changes/h
- Method of particle size determination: TSI Aerodynamic Particle Sizer


TEST ATMOSPHERE
- Brief description of analytical method used: At least three times a day by HPLC.
- Samples taken from breathing zone: Yes


VAPOR GENERATION
- Apparatus: Syringe pump (Sage Instruments, Cambridge, MA)
- Method: Liquid test material was introduced at the top of the evaporator into a spiral groove of the inner wall and allowed to flow downward

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Reverse phase HPLC was performed using 501 Waters liquid chromatograph equipped with an SP 8400 ultraviolet/visible detector and an SP 4290 Computing Integrator.
- Detection limit: 0.014 ppm

Duration of treatment / exposure:

6 h/d, 5 d/wk for 13 wk, and for the 2 d during the 14 wk

Frequency of treatment:

Daily (6 h/d), 5 d/wk

Remarks:

Doses / Concentrations:
0, 0.31 ± 0.061, 0.72 ± 0.096 and 1.46 ± 0.146 ppm
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
0 (control), 0.4, 0.8 and 1.6 ppm
Basis:
other: target conc.

No. of animals per sex per dose:

Ten

Control animals:

yes

Details on study design:

- Rationale for animal assignment: Animals were assigned by computer-based randomization program

Positive control:

No data

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At the time of body weight collection and just preceding sacrifice


BODY WEIGHT: Yes
- Time schedule for examinations: Pre-exposure, once a wk during exposure and immediately preceding sacrifice


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to first exposure and following each exposure
- Dose groups that were examined: All


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to terminal sacrifice
- Anaesthetic used for blood collection: Yes (Methoxyflurane)
- Animals fasted: Yes
- How many animals: All


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to terminal sacrifice
- Animals fasted: Yes
- How many animals: All


URINALYSIS: Yes
- Time schedule for collection of urine: At the end of the exposure regimen
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see Appendix 4)
- Organ weights: Brain, liver, kidneys, lungs and adrenal glands from all surviving animals and testis from male

HISTOPATHOLOGY: Yes (see Appendix 4) (including epididymes, prostate, testes, uterus and ovaries)

Statistics:

Bartlett's test for homogeneity of variances, ANOVA, Duncan's multiple range test or t- test

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

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:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
- Clinical signs: Respiratory difficulties (gasping, audible respiration), reddening of the ears and paws
- Mortality: 15 % in 1.6 ppm group of rats (males only)

BODY WEIGHT AND WEIGHT GAIN
Decreased body weight gain in 0.4, 0.8 and 1.6 ppm groups

OPHTHALMOSCOPIC EXAMINATION
No changes

HAEMATOLOGY
Increased mean corpuscular volume (males) and erythrocyte count (females) in 0.4, 0.8 and 1.6 ppm group rats

CLINICAL CHEMISTRY
Decreased albumin (0.8 and 0.4 ppm) and glucose concentration (0.4 ppm) in females

URINALYSIS
Decreases in the urine volume for the 0.8 and 1.6 ppm group females

ORGAN WEIGHTS
Increased absolute and relative lung weights in 1.6 ppm group

GROSS PATHOLOGY
Color change and the presence of emphysematous lungs in the 1.6 ppm group

HISTOPATHOLOGY: NON-NEOPLASTIC
- Necrosis, ulceration, squamous metaplasia, and inflammatory changes occurred in the nasal cavity
- Necrosis and inflammatory changes in the trachea and larynx
- Hemorrhage, edema, necrosis, submucosal fibrosis and fibrous polyps, which projected into the lumen, in the lung
- Squamous metaplasia and inflammatory changes throughout the respiratory tract, with bronchiolar submucosal fibrosis in the lungs (at 1.6 ppm)
- Squamous metaplasia and inflammatory infiltrates in upper respiratory tract (at 0.4 and 1.6 ppm), bronchiolar submucosal fibrosis (at 0.8 ppm)

Key result

Dose descriptor:

NOAEC

Remarks on result:

not determinable

Remarks:

no NOAEC identified

Key result

Dose descriptor:

LOAEC

Effect level:

0.4 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Effects observed at all dose levels: Clinical signs; mortality; body weight; haematology; clinical chemistry; urinalysis; gross pathology; organ weights; histopathology

Key result

Critical effects observed:

not specified

Conclusions:

Under the test conditions, rats exposed to vapor of the test material had evidence of toxicity at all exposure concentrations. A NOAEC could not be established, although the incidence, severity and depth of histological lesions within the respiratory tract generally decreased with decreasing exposure concentration. The LOAEC for the study can be considered to be 0.4 ppm, the lowest dose tested.

Executive summary:

A study was conducted to evaluate the effects of 14 weeks of repeated exposure to vapour of the test substance in rats according to a method equivalent or similar to OECD Guideline 413, in compliance with GLP. Sprague-Dawley rats (10/sex/group) received whole-body exposures for 6 h/d, 5 d/week, for 13 week to either filtered air or test substance vapour at target concentrations of 0 (control), 0.4, 0.8 or 1.6 ppm. All animals were monitored for toxic effects including clinical observations, body and organ weights, haematology, serum chemistry, urinalysis evaluations, ophthalmic examinations, and macroscopic and microscopic evaluations. Mean analytical concentrations of 0.31, 0.72 and 1.46 ppm of test substance were obtained. The incidence of mortality at 1.6 ppm was 15%. Respiratory difficulty and reddening of ears and paws were observed at 0.8 and 1.6 ppm. Effects on body weight gain were generally concentration-related, being depressed at 1.6 ppm and sporadically depressed at 0.8 and 0.4 ppm. Many of the haematology and serum chemistry parameters were abnormal at 1.6 ppm. Increased mean corpuscular volume and erythrocyte count, and decreased albumin concentration, glucose concentration and urine volume were observed at 0.4 or 0.8 ppm. At necropsy, the principal observations included a colour change (congestion) and emphysematous lungs at 1.6 ppm. Absolute and/or relative lung weight values were increased for rats at 1.6 ppm. In the nasal cavity, lesions included necrosis, ulceration, squamous metaplasia and inflammatory changes; necrosis and inflammatory changes generally occurred in the larynx and trachea; pulmonary changes included congestion, haemorrhage, necrosis, inflammation and in several of the animals, bronchiolar submucosal fibrosis. At 1.6 ppm, squamous metaplasia and inflammatory changes were observed throughout the respiratory tract, with bronchiolar submucosal fibrosis. At 0.4 and 0.8 ppm, exposure-related lesions were principally found in upper respiratory tract and involved squamous metaplasia and inflammation. Under the test conditions, rats exposed to vapour of the test substance had evidence of toxicity at all exposure concentrations. A NOAEC was not established, although the incidence, severity and depth of histological lesions within the respiratory tract generally decreased with decreasing exposure concentration. The LOAEC for the study can be considered to be 0.4 ppm, the lowest dose tested (Klonne, 1990).

Reference 3

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Charles River Breeding Laboratories, New York 12484
- Age at study initiation: Male: 8 wk, female: 9 wk
- Weight at study initiation: Male: 304-336 g; Female: 199-235 g
- Housing: Individually housed in stainless steel wire mesh cages
- Diet (e.g. ad libitum): Purina Certified Rodent Chow #5002, ad libitum
- Water (e.g. ad libitum): Automated watering system, ad libitum
- Acclimation period: 5 wk

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-70 %
- Photoperiod (hrs dark / hrs light): 12h dark/12 h light

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

not specified

Vehicle:

other: acetone

Remarks on MMAD:

MMAD / GSD: MMAD: 0.9-1.5 microns
GSD: 1.5-1.9

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Glass and stainless steel exposure chambers
- Source and rate of air: Matheson pressure gauze, Nupro metering valve and Dwyer flowmeter
- System of generating particulates/aerosols: Atomizing nozzle (Spraying Systems Co.; 1/4" JSS nozzle with a #1A spray set-up) with glass syringe mounted in a Sage Syringe Pump (Model #352 or #341)
- Method of particle size determination: TSI Aerodynamic Particle sizer equipped with Diluter
- Air flow rate: 200-212 L/min
- Air change rate: 4.7-5.0 min


TEST ATMOSPHERE
- Brief description of analytical method used: Samples collected using impingers and analysed by HPLC
- Samples taken from breathing zone: Yes


VEHICLE
- Composition of vehicle: Acetone
- Lot/batch no. of vehicle: 856554

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Analysis of test material in impingers by liquid chromatography
- Limit of detection: 2.78 ng injected (50 µL of a 0.0555 µg/mL standard)

Duration of treatment / exposure:

6 h/d, 5d/wk for 4 wk

Frequency of treatment:

Daily (6 h/d), 5 d/wk

Remarks:

Doses / Concentrations:
0, 0.38, 1.5 and 4.4 mg/m3
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
0, 0.5, 1.5 and 5.0 mg/m3
Basis:
other: Target conc.

No. of animals per sex per dose:

Five

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment: Ramdomly assigned by computerized randomization

Positive control:

Not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly


BODY WEIGHT: Yes
- Time schedule for examinations: Once pretest and on Day 1, 5, 8, 12, 15, 19, 22, 26 and 29


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 29
- How many animals: 5/sex/group
- Parameters examined: Hemoglobin concentration, hematocrit, erythrocyte count, clotting time, total and differential leukocyte counts

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: No
- How many animals: 5/sex/group
- Parameters examined: Serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT); blood urea, nitrogen, creatinine, fasting glucose, total protein, sodium, potassium, chloride, calcium and inorganic phosphorus

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes (including gonads, ovaries and testes with epididymes)

Statistics:

Anova, Bartletts, Dunnets, Kruskal- Wallis, Regression analysis, Jonckheere's statistic and Summed Rank Test

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
- Clinical signs: Decrease in the activity at all dose levels, increased salivation at 1.5 and 5 mg/m3 and closed eyes at 5 mg/m3
- Mortality: None


BODY WEIGHT AND WEIGHT GAIN
No significant changes in body weight except decreased body weight gain in males at 5.0 mg/m3


HAEMATOLOGY
No treatment-related effects


CLINICAL CHEMISTRY
Significant elevation in serum calcium and phosphorous in males at 5 mg/m3


ORGAN WEIGHT
Increased relative lung weight in females at 5 mg/m3


GROSS PATHOLOGY/ HISTOPATHOLOGY (NON-NEOPLASTIC)
- Evidence of discoloured lungs (red foci) at 0.5 (in 1 male), 1.5 (in 1 male and 1 female) and 5 mg/m3 (in 4 males)
- Microscopically, the significant findings were subacute/chronic inflammation in the lungs and appearance of hyperplastic and metaplastic changes in the bronchi at 5 mg/m3

Key result

Dose descriptor:

NOAEC

Effect level:

1.5 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Key result

Critical effects observed:

not specified

Conclusions:

Under the test conditions, the NOAEC of the test material was considered to be 1.5 mg/m3 in rat.

Executive summary:

A study was conducted to evaluate the inhalation toxicity of the test substance in rat according to a method equivalent or similar to OECD Guideline 412, in compliance with GLP. The test substance as a respirable aerosol was administered by inhalation to Sprague-Dawley rats (5/sex/group) for 6 h/d, 5 d/week, for 4 weeks at target concentrations of 0, 0.5, 1.5 and 5.0 mg/m3 (measured concentrations of 0 (acetone vapour), 0.38, 1.5 and 4.4 mg/m3). Animals were given detailed physical examinations. Body weight, organ weights, haematology and clinical chemistry were observed and histopathological examinations were conducted. Increased salivation, closed eyes, decreased body weight gain and elevated serum calcium/phosphorus levels (in males), increased relative lung weight (in females) and pathological changes in lungs (subacute/chronic inflammation in the lungs and appearance of hyperplastic and metaplastic changes in the bronchi) were observed at 5.0 mg/m3. Under the study conditions, the NOAEC of the test substance was considered to be 1.5 mg/m3 in rat (Rinehart, 1987).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

4 mg/m³

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral

A repeated dose reproductive/developmental toxicity study was conducted to screen potential adverse effect of test substance on systemic toxicity and reproduction, including embryo/foetal development in the rat according to OECD Guideline 421, in compliance with GLP. The test substance was administered by gavage at concentrations of 0, 15, 150 and 250 mg/kg bw/day to groups of ten rats of either sex for a period of 19 d in males and 40 -41 d in females. On Day 5 post-partum, all surviving animals were killed and examined macroscopically for internal and external abnormalities. All adults and offspring were observed for clinical signs. Male body weights and food consumption per cage were recorded weekly throughout the study. Body weights and food consumption per cage of females were recorded weekly until mating. After mating, body weights of females were recorded on Gestation Days (GD) 0, 7, 14, and 20 and on Lactation Days (LD) 1 and 4, and food consumption of females was determined for GD 1-7, 7-14 and 14-20. Female food consumption also was recorded for the period covering LD 1-4. The litter signs and individual pup bodyweights were recorded on Days 1 and 4 post-partum. Post mortem macroscopic examinations were performed on all adults and offspring including decedents. Histopathology was carried out on reproductive organs from control and high dose group parental animals at termination. There was a total of 13 adult mortalities, distributed across all dose groups. Twelve of these were associated with dosing trauma and the nature of the test substance. The distribution of the mortalities suggested that it was not a consequence of systemic toxicity. At 250 mg/kg bw/day, there were clinical signs of reaction to a potentially irritating substance. These included increased salivation pre- and post-dosing, evidence of a reduction in bodyweight gain throughout the different phases of the study and significant reductions in food consumption. Post-mortem evaluation at termination showed limited evidence of effects on the gastro-intestinal tract. At 150 mg/kg bw/day, there were similar clinical signs of reaction to dosing and the post-mortem evaluation of adults at termination showed isolated incidents of potential gastro-intestinal effects. There were however no significant effects on bodyweight and food consumption during the in-life phase of the study. At 15 mg/kg bw/day, there were similar clinical signs of increased salivation. There were no significant findings at post-mortem macroscopic examination and no effects upon bodyweight or food consumption during the course of the study. There were no significant histopathological changes observed for the reproductive organs of adults at termination in any dose group. Under the study conditions, administration of the test substance to adult male and female rats throughout maturation, mating, gestation and early lactation resulted in significant effects at 250 mg/kg bw/day. Although effects were seen at 150 mg/kg bw/day, they were not considered to be toxicologically significant and this dose was therefore considered to be the NOAEL (Knox, 2005).

Inhalation

A study was conducted to evaluate the inhalation toxicity of the test substance in rat according to a method equivalent or similar to OECD Guideline 412, in compliance with GLP. The test substance as a respirable aerosol was administered by inhalation to Sprague-Dawley rats (5/sex/group) for 6 h/d, 5 d/week, for 4 weeks at target concentrations of 0, 0.5, 1.5 and 5.0 mg/m3 (measured concentrations of 0 (acetone vapour), 0.38, 1.5 and 4.4 mg/m3). Animals were given detailed physical examinations. Body weight, organ weights, haematology and clinical chemistry were observed and histopathological examinations were conducted. Increased salivation, closed eyes, decreased body weight gain and elevated serum calcium/phosphorus levels (in males), increased relative lung weight (in females) and pathological changes in lungs (subacute/chronic inflammation in the lungs and appearance of hyperplastic and metaplastic changes in the bronchi) were observed at 5.0 mg/m3. Under the study conditions, the NOAEC of the test substance was considered to be 1.5 mg/m3 in rat (Rinehart, 1987).

A study was conducted to evaluate the effects of 14 weeks of repeated exposure to vapour of the test substance in rats according to a method equivalent or similar to OECD Guideline 413, in compliance with GLP. Sprague-Dawley rats (10/sex/group) received whole-body exposures for 6 h/d, 5 d/week, for 13 week to either filtered air or test substance vapour at target concentrations of 0 (control), 0.4, 0.8 or 1.6 ppm. All animals were monitored for toxic effects including clinical observations, body and organ weights, haematology, serum chemistry, urinalysis evaluations, ophthalmic examinations, and macroscopic and microscopic evaluations. Mean analytical concentrations of 0.31, 0.72 and 1.46 ppm of test substance were obtained. The incidence of mortality at 1.6 ppm was 15%. Respiratory difficulty and reddening of ears and paws were observed at 0.8 and 1.6 ppm. Effects on body weight gain were generally concentration-related, being depressed at 1.6 ppm and sporadically depressed at 0.8 and 0.4 ppm. Many of the haematology and serum chemistry parameters were abnormal at 1.6 ppm. Increased mean corpuscular volume and erythrocyte count, and decreased albumin concentration, glucose concentration and urine volume were observed at 0.4 or 0.8 ppm. At necropsy, the principal observations included a colour change (congestion) and emphysematous lungs at 1.6 ppm. Absolute and/or relative lung weight values were increased for rats at 1.6 ppm. In the nasal cavity, lesions included necrosis, ulceration, squamous metaplasia and inflammatory changes; necrosis and inflammatory changes generally occurred in the larynx and trachea; pulmonary changes included congestion, haemorrhage, necrosis, inflammation and in several of the animals, bronchiolar submucosal fibrosis. At 1.6 ppm, squamous metaplasia and inflammatory changes were observed throughout the respiratory tract, with bronchiolar submucosal fibrosis. At 0.4 and 0.8 ppm, exposure-related lesions were principally found in upper respiratory tract and involved squamous metaplasia and inflammation. Under the test conditions, rats exposed to vapour of the test substance had evidence of toxicity at all exposure concentrations. A NOAEC was not established, although the incidence, severity and depth of histological lesions within the respiratory tract generally decreased with decreasing exposure concentration. The LOAEC for the study can be considered to be 0.4 ppm, the lowest dose tested (Klonne, 1990).

A study was conducted to evaluate the toxic effects of 14 weeks of repeated exposure to vapour from test substance in mice. The study was performed according to OECD Guideline 413, in compliance with GLP. CD-1 mice(10/sex/group) received whole-body exposures for 6 h/d, 5 d/week, for 14 weeks to either filtered air or test substance vapour at target concentrations of 0 (control), 0.4, 0.8 or 1.6 ppm. All animals were monitored for toxic effects including clinical observations, body and organ weights, haematology, serum chemistry, urinalysis evaluations, ophthalmic changes, and macroscopic and microscopic evaluations. Mean analytical concentrations of 0.31, 0.72 and 1.46 ppm of test substance were obtained. Respiratory difficulty (e.g. gasping), reddening of the ears and paws, blepharospasm and alopecia were observed in mice at 0.8 and 1.6 ppm. The incidence of mortality was 5, 20, and 80% for the 0.4, 0.8, and 1.6 ppm groups, respectively. Effects on body weight gain were generally concentration related, being depressed at 1.6 ppm and sporadically depressed at 0.8 and 0.4 ppm. No concentration-related changes in haematology, serum chemistry and urinalysis parameters were observed. At necropsy, the principal changes included pulmonary congestion and alopecia. Biologically- significant organ weight changes in the lungs, absolute and/or relative lung weight values were increased for female mice at 0.8 ppm. In the nasal cavity, the lesions included necrosis, ulceration, squamous metaplasia and inflammatory changes; necrosis and inflammatory changes generally occurred in the larynx and trachea; pulmonary changes included congestion, haemorrhage, necrosis, inflammation and in several of the animals, bronchiolar submucosal fibrosis. Inflammatory changes and fibrosis in the lungs were seen at all dose levels. Under the test conditions, mice exposed to vapor of test substance had evidence of toxicity at all exposure concentrations. A NOEC could not be established, although the incidence, severity and depth of histological lesions within the respiratory tract generally decreased with decreasing exposure concentration. The LOAEC for the study can be considered to be 0.4 ppm, the lowest dose tested (Klonne, 1990).

Overall assessment

In both oral and inhalation studies, repeated exposure to the test substance caused effects that could be mainly linked to its irritating properties. No significant direct systemic effects were observed. As a result, no systemic DNEL values will be calculated in the present risk assessment.

Justification for classification or non-classification

With an oral NOAEL of 150 mg/kg bw/day, m-TMXDI does not meet the criteria for repeated dose classification via this route. Based on the effects seen in the 90 d inhalation studies, m-TMXDI meets the criteria for classification as STOT Rep. Exp. 1 - H372: Causes damage to organs <respiratory system> through prolonged or repeated exposure <inhalation> according to CLP (EC 1272/2008) criteria.