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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
repeated dose toxicity: inhalation
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with OECD GLP (1997) regulations.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 421 (Reproduction/Developmental Toxicity Screening Test)
Deviations:
yes
Principles of method if other than guideline:
Deviations: The temperature of the exposure chamber during exposure was above 24 C incidentally on a few days for all groups. Food consumption was not determined during the first week of the post-mating period, due to prolongation of the mating period. Testis and epididymides weight were determined for all animals instead of 5 animals per group. Lung weight was determined for all animals and organ weights were erroneously not determined for animal number 82. Reproductive organs in the mid concentration animals instead of the high concentration animals were examined by histopathology, since the high concentration group was stopped preterm. These deviations were considered not to have affected the validity of the study.
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Liquid
Details on test material:
- Name of test material (as cited in study report): MTDID 948
- Substance type: Mono-constituent
- Physical state: Liquid
- Analytical purity: 94.4%
- Purity test date: 16 April, 2014
- Lot/batch No.: 20014, Unit 027
- Expiration date of the lot/batch: 11 August, 2016
- Storage condition of test material: Ambient temperature

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories
- Age at study initiation: 7-9 weeks
- Weight at study initiation: (P) Male means: 330.2-337.34 g; Female means: 207.31-209.29 g; (F1) Male means: 6.24-6.67 g; Female means: 4.39-5.25 g
- Fasting period before study: No data, fasting was conducted before blood draws
- Housing: Animals were housed in macrolon cages with a bedding of wood shavings, strips of paper and a wooden block for enrichment. After allocation, the animals were housed four or five rats to a cage, separated by sex. For mating, one male and one female were housed together. Mated females were housed individually in macrolon cages.
- Diet (e.g. ad libitum): Special Diet Services VRF-1 (FG) ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: 13 Days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 18 March, 2015 To: 11 May, 2015

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
nose only
Vehicle:
clean air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:Aluminum or polypropylene inhalation chamber (46.7 L).
- Method of holding animals in test chamber: Animals were held in Battelle plastic animal holders in rodent tube sections with 20 exposure ports
- Source and rate of air: At least 1 L/min per animal.
- Temperature, humidity, pressure in air chamber: 29-25 C, 30-70% humidity

TEST ATMOSPHERE
- Brief description of analytical method used: Test atmosphere concentration was measured by total carbon analysis (Sick Maihak EuroFID total carbon analyzer).
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test atmosphere concentration was measured by total carbon analysis (Sick Maihak EuroFID total carbon analyzer).
Duration of treatment / exposure:
6 hours a day
Frequency of treatment:
Premating period: Male and female animals were exposed during 2 weeks prior to mating to the test substance for 5 days/week (10 exposure days in total).
Mating period: Male and female rats were exposed daily.
Gestation period: Female animals were exposed daily from mating (finding sperm positive vaginal smear; gestation day 0) up to (and including) gestation day 19. Females that appeared to be not pregnant were exposed daily during the same period. Male rats were exposed daily until sacrifice on 1 May 2015.
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (control), 150, 350, or 750 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
12
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: A 14 day range-finding study was conducted prior to this study.
- Rationale for animal assignment: Random
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed aily in the morning hours by cage-side observations and, if necessary handled to detect signs of toxicity. The animals were also observed halfway through the 6-hour exposure period, in particular to monitor any breathing abnormalities and restlessness. All animals were also thoroughly checked again after exposure.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily in the morning and after exposure.

BODY WEIGHT: Yes
- Time schedule for examinations: Prior to the study and twice weekly thereafter.

FOOD CONSUMPTION:
- Food consumption: Yes, measured per cage over the same periods as the body weights were measured, except during the mating period. The results were expressed in g/animal/day and g/kg bw/day
Sacrifice and pathology:
SACRIFICE
- Male animals: All surviving animals were sacrificed after a total of 25 exposure days (1 May 2015).
- Maternal animals: All surviving animals were sacrified at least four days after delivery. Females that failed to mate were sacrifieced at least 24 days after the last mating date.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
Other examinations:
For each mating, the following data were presented for each group: Number of females mated, number of males mated, number of females inseminated, number of males with pregnant females, number of pregnant females (confirmed by presense of implantation sites at necropsy), number of females surviving delivery, number of females with liveborn and stillborn pups, number of pups delivered, number of corpora lutea, number of implantation sites, litter size (mean number of pups delivered).

The following parameters were calculated: Pre-coital time, duration of gestation female mating index, male fertility index, female fertility index, gestation index, live birth index, sex ratio day 0 or 4, prenatal loss, perinatal loss.

Fpr each mating the following data were presented for each group: Number of live pups at day 0 or 4, number of pups lost at day 4, number of litters lost entirely, number of (male/female) pups at day 0 or 4,

The following parameters were calculated: Viability (lactation index) day 0-4
Statistics:
Data were analyzed using the methods below. Tests were generally performed as two-sided tests with results taken as significant where the probability of the results was P<0.05 or P<0.01.
-Functional observation battery: one-way analysis of variance followed by Dunnett's multiple comparison tests (continuous data), Kruskal-Wallis non-parametric analysis of variance followed by multiple comparison tests (rank order data) or Pearson chi-square analysis (categorical data).
-Motor activity data: total distance moved: one-way analysis of variance followed by Dunnett's multiple comparison tests; habituation of activity: repated measures analysis of variance on time blocks (each session consists of 5 time blocks of 6 minutes each).
-Incidences of histopathological changes: Fisher's exact probability test.

Results and discussion

Results of examinations

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:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): 750 ppm animals exhibited hunched posture, piloerection, appeared thin, had muscle weakness and encrustations of the eyes. Incidence and severity of signs increased over two weeks of exposure. One female in the high-dose group (750 ppm) was found dead on Day 4 without the preceding clinical observations. One female in the 750 ppm group was found dead on Day 18. One male animals in the 750 ppm group was sacrificed moribund on Day 22. Based on the deaths, clinical signs and body weight loss observed in the 750 ppm dose group animals, it was decided to discontinue exposure for the group from Day 23 onwards. All remaining animals in the 750 ppm group were sacrificed on Day 24. No abnormal clinical signs or mortaility was observed in other dose groups.

BODY WEIGHT AND WEIGHT GAIN: Males in the 750 ppm group showed progressing body weight loss from the start of exposure onwards. Mean body weights were statistically significantly lower at the end of premating and during post-mating. Body weight changes were statistically significantly different in all study intervals. Females in the 750 ppm group showed a more limited decrease in body weight gain, although weight changes were statistically significantly decreased at the end of the pre-mating period. There were no statistically significant differences in body weights of the 150 or 350 ppm animals compared to controls.

FOOD CONSUMPTION: Females in the 750 ppm group showed slightly lower food consumption in the premating phase, which was statistically significant during the second week of premating. No treatment-related changes in food consumption effects were noted in the 150 and 350 ppm animals.

HAEMATOLOGY: Not examined

CLINICAL CHEMISTRY: Not examined

ORGAN WEIGHTS (PARENTAL ANIMALS): As the 750 ppm group was discontinued, organ weights are not comparable to the control group. in the 150 and 350 ppm groups, there were no significant changes in the weight of male reproductive organs and the liver. Relative lung weight was slightly, but statistically significantly increased in both males and females in the 350 ppm group. In absence of a satistically significant differnce in absolute lung weight, this effect was considered non-adverse.

GROSS PATHOLOGY (PARENTAL ANIMALS): 750 ppm males sacrificed early revealed three males with small prostate glands, four males with small seminal vesicles, four males with cryptochidism, and one male with small testes and epididymis. Three males and one female in the 750 ppm group had pale kidneys. Two males and two females in the 750 ppm group had a small thymus and one male had a small spleen. No abnormal findings were observed in the 150 and 350 ppm groups upon gross necropsy.

HISTOPATHOLOGY (PARENTAL ANIMALS): Microscopic analysis of the male animals revealed minimal unilateral seminiferous tubular atrophy in both the control group (two animals) and the 350 ppm group (three animals). In addition two animals of the 350 ppm group showed mild seminiferous tubular atrophy. In agreement with this observation, minimal unilateral cellular debris was observed in the epididymis of two animals of both the control group and the 350 ppm group. In addition, three animals of the 350 ppm group showed mild cellular debris in the epididymis. No statistical significance was reached for these findings in treatment groups compared to controls. Unilateral, a sperm granuloma was found in the epididymis of an animal of the 350 ppm group. The liver of one male animal of the 350 ppm group showed multifocal hepatocellular degeneration and hemorrhages.

Effect levels

Dose descriptor:
NOAEC
Effect level:
ca. 350 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: NOAEC set based on absence of effects on clinical signs, body weight, food consumption and the slight effect on relative lung weight.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the results of the study, the parental No Observed Adverse Effect Concentration (NOAEC) of the test article is 350 ppm (4.29 mg/L, vapor).
Executive summary:

The repeat dose toxicity and reproductive and developmental toxicity potential of the test article was evaluated in male and female Wistar rats. The study was conducted in compliance with OECD GLP (1997) regulations. The test method was based on OECD 421 (1995). The test article was administered by inhalation at 0 (control), 150, 350, and 750 ppm (nose only, 6 hours per exposure per day) to groups of 12 male and 12 female rats during a premating period of 2 weeks and during mating and gestation up to gestation day 19. Male animals were sacrificed after a total of 25 exposure days. Dams and pups were sacrificed four or five days after delivery. In-life parameters recorded included clinical signs, body weight, food consumption, mating parameters, gestational and parturition parameters and litter parameters. At necropsy, testes and epididymides were weighed for male animals and liver and lung weight was determined for all animals. Liver and respiratory tract were preserved for all animals but not examined histopathologically. Reproductive organs were examined histopathologically for animals of the mid exposure group (350 ppm) and the control group. Animals exposed to 750 ppm exhibited hunched posture, piloerection, appeared thin, had muscle weakness and encrustations of the eyes. Incidence and severity of signs increased over two weeks of exposure. One female in the high-exposure group (750 ppm) was found dead on Day 4 without the preceding clinical observations. One female in the 750 ppm group was found dead on Day 18. One male animal in the 750 ppm group was sacrificed moribund on Day 22. Based on the early mortalities, clinical signs and body weight loss observed in the 750 ppm group animals, it was decided to discontinue exposure for the group from Day 23 onwards. All remaining animals in the 750 ppm group were sacrificed on Day 24. No abnormal clinical signs or mortality was observed in other exposure groups. Males in the 750 ppm group showed progressing body weight loss from the start of exposure onwards and mean body weights were statistically significantly lower at the end of premating and during post-mating. Females in the 750 ppm group showed a more limited decrease in body weight gain, although weight changes were statistically decreased at the end of the pre-mating period. There were no statistically significant differences in body weights of the 150 or 350 ppm animals compared to controls. In the 750 ppm group, 11 females were places with males. Due to the unscheduled death of one male and one female during mating and the deteriorating conditions of the other animals in this group, exposure was discontinued and the animals were sacrificed after 9 days of cohabitation. At the time of unscheduled sacrifice, 4 out of 11 females were sperm-positive and considered mated. In the control, 150 and 350 groups, 12 females were placed with males for mating. In the control group, one female was not mated and another female did not deliver, resulting in 10 litters. In the 150 ppm and 350 ppm groups, all females were mated and this resulted in 12 litters in the 150 ppm group and 11 litters in the 350 ppm group. There were no relevant differences in pre-coital time, mating index, male or female fertility index between the 150 ppm, 350 ppm and control groups and no treatment-related effects were observed in the mean number of corpora lutea and the mean number of implantation sties. Reproductive performance was not affected by treatment. In each group, the duration of gestation was comparable. The mean number of pups was comparable in all groups and there were no stillborn pups. As the 750 ppm group was discontinued, organ weights are not comparable to the control group. In the 150 and 350 ppm groups, there were no significant changes in the weight of male reproductive organs and the liver. Relative lung weight was slightly, but statistically significantly increased in both males and females in the 350 ppm group. In absence of a statistically significant difference in absolute lung weight, this effect was considered non-adverse. Males from the 750 ppm group that were sacrificed early revealed three animals with small prostate glands, four with small seminal vesicles, four with cryptochidism, and one with small testes and epididymis. Three males and one female in the 750 ppm group had pale kidneys. Two males and two females in the 750 ppm group had a small thymus and one male had a small spleen. No abnormal findings were observed in the 150 and 350 ppm groups upon gross necropsy. Microscopic analysis of the male animals revealed minimal unilateral seminiferous tubular atrophy in both the control group (two animals) and the 350 ppm group (three animals). In addition two animals of the 350 ppm group showed mild seminiferous tubular atrophy. In agreement with this observation, minimal unilateral cellular debris was observed in the epididymis of two animals of both the control group and the 350 ppm group. In addition, three animals of the 350 ppm group showed mild cellular debris in the epididymis. No statistical significance was reached for these findings in treatment groups compared to controls. A Unilateral sperm granuloma was found in the epididymis of an animal of the 350 ppm group. The liver of one male animal of the 350 ppm group showed multifocal hepatocellular degeneration and hemorrhages. The number of pups born and the sex ratio were comparable between the treatment groups and controls. The viability index was not affected by treatment. The viability index was 99% in the control group, 98.3% in the 150 ppm group, 89.9% in the 350 ppm group. There were no treatment-related signs in pups during the lactation period. There were no differences in mean pup weight between the test groups and the controls on Day 0 or Day 4 or lactation. No gross abnormalities were observed in any pups when examined externally. Based on the results of the study, the parental No Observed Adverse Effect Concentration (NOAEC) of the test article is 350 ppm (4.29 mg/L, vapor).