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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1992 - 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Methacrylonitrile
EC Number:
204-817-5
EC Name:
Methacrylonitrile
Cas Number:
126-98-7
Molecular formula:
C4H5N
IUPAC Name:
methacrylonitrile
Details on test material:
- Name of test material (as cited in study report): Methacrylonitrile
- Substance type: organic aliphatic nitrile
- Physical state: liquid, colorless
- Analytical purity: 99.9%
- Lot/batch No.: JY00427ET
- Supplier: Aldrich Chemical Company, Milwaukee, WI
- Stability under test conditions: no significant decomposition was observed by gas chromatographical analysis during the study
- Storage condition of test material: protected from light at 5 degrees Centigrade

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, NY and Portage, MI
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 116-123g (males), 105-110g (females)
- Housing: 5 animals per polycarbonate cage with Sani-Chip hardwood chips (P.J. Murphy Forest Products Corp., Montville, NJ) as bedding changed twice weekly, spun bounded DuPont 2024 polyester cage filters (Snow filtration co., Cincinnati, OH), and stainless steel racks.
- Diet (e.g. ad libitum): pelleted NIH-07 open formula diet (Zeigler Brothers, Inc., Gardners, PA) ad libitum
- Water (e.g. ad libitum): from Columbus Municipal Supply available ad libitum via automatic watering system
- Acclimation period: 11-15 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20.6-23.9 degrees Centigrade
- Humidity: 35-65 %
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1992-07-13 To: 1992-10-16

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Dose formulations were prepared by mixing methacrylonitrile with deionized, purified water to give the required concentrations. Dose formulations were prepared approximately every 4 weeks during the studies. Dose formulations used in the 13-week studies were stored in amber-glass bottles at room temperature.

VEHICLE
- Amount of vehicle (if gavage): dosing volume: 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability studies conducted by the study laboratory with gas chromatography indicated that a 0.015 mg/mL formulation was stable for 35 days when stored protected from light at 5 degrees Centigrade or at room temperature.

At the study laboratory, dose formulations prepared at the beginning, midpoint, and end of the 13-week studies were analyzed with gas chromatography prior to dosing; all formulations administered to animals were within 10 % of target concentrations. However, analyses of animal room samples prepared at the beginning and midpoint of the studies indicated significant losses of methacrylonitrile during administration and storage; on average, the concentrations of methacrylonitrile in these formulations were 20 % less than the targeted concentrations. Because of these losses, dosing procedures were modified to ensure that dose formulation containers were open for a minimum amount of time during dosing and were thoroughly sealed after dosing. Animal room sample analyses of formulations handled under these conditions indicated minimal chemical losses
ranging from 5 % to 10 %.
Duration of treatment / exposure:
Animals were treated for 13 weeks exept for the interim evaluation which was conducted after 32 days.
Frequency of treatment:
5 times per week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 7.5, 15, 30, 60, or 120 mg methacrylonitrile/kg body weight
Basis:
actual ingested
No. of animals per sex per dose:
In the 13-week studies, 20 male and 20 female rats and mice per dose group were treated. Ten animals from each group were pre-selected for interim
evaluations; these animals were dosed for 32 days and then killed and examined.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose selection for the 13-week studies was based on doses in the 14-day drinking water study which caused high mortality and clinical findings of acute central nervous system toxicity and effects similar to those caused by cyanide. Furthermore, dose selection was also based on the reported gavage LD50 values of 200 mg/kg for rats (Pozzani et al., 1968).
- Rationale for animal assignment (if not random): Animals were distributed randomly into groups of approximately equal initial mean body weights.
Positive control:
No positive control was performed.

Examinations

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: weekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the 13 week treatment and the 32 day interim phase. Additionally, the 32-day interim evaluation rats were also evaluated after 4 days of dosing.
- Anaesthetic used for blood collection: Yes: CO2/O2 gas mixture
- Animals fasted: No data
- How many animals: all
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the 13 week treatment and the 32 day interim phase. Additionally, the 32-day interim evaluation rats were also evaluated after 4 days of dosing.
- Anaesthetic used for blood collection: Yes: CO2/O2 gas mixture
- Animals fasted: No data
- How many animals: all
- Parameters checked in table 1 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 2)
HISTOPATHOLOGY: Yes (see table 2)
Other examinations:
At the end of the 13-week studies, sperm motility and vaginal cytology evaluations were performed on male rats in the 0, 15, 30, and 60 mg/kg groups, female rats in the 0, 30, 60, and 120 mg/kg groups. Males were evaluated for necropsy body and reproductive tissue weights, spermatid data, and epididymal spermatozoal data. Females were evaluated for necropsy body weight, estrous cycle length, and the percentage of cycle spent in the various stages.
Statistics:
The incidences of lesions are reported as the numbers of animals bearing such lesions at a specific anatomic site and the numbers of animals with that site examined microscopically. The Fisher exact test, a procedure based on the overall proportion of affected animals, was used to determine significance.

Prior to analysis, extreme values identified by the outlier test of Dixon and Massey (1951) were examined by NTP personnel.

Two approaches were employed to assess the significance of pairwise comparisons between dosed and control groups in the analysis of continuous variables. Organ and body weight data, which have approximately normal distributions, were analyzed with the parametric multiple comparisons procedures of Williams (1971, 1972) and Dunnett (1955).

Clinical chemistry, hematology, spermatid, and spermatozoal data, which typically have skewed distributions, were analyzed with the nonparametric multiple comparison methods of Shirley (1977) and Dunn (1964).

Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of dose-response trends and to determine whether a trend-sensitive test (Williams. or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose response (Dunnett’s or Dunn’s test). If the P value from Jonckheere’s test was greater than or equal to 0.10, Dunn’s or Dunnett’s test was used rather than Shirley’s or William’s test.

Average severity values were analyzed for significance using the Mann-Whitney U test (Hollander and Wolfe, 1973).

Because vaginal cytology data are proportions (the proportion of the observation period that an animal was in a given estrous stage), an arcsine transformation was used to bring the data into closer conformance with normality assumptions.

Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for the simultaneous equality of measurements across dose levels.

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):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
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:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY
Among groups scheduled for the 32-day interim evaluation, 9 of 10 males in the 120 mg/kg bw group died during the first week; all female rats survived. Clinical findings of toxicity at the 32-day interim evaluation were dose dependent and included lethargy, lacrimation, tremors, convulsions, ataxia, and abnormal breathing. These effects were observed within minutes of dosing and disappeared within several hours after dosing.

Among groups scheduled for evaluation at the end of the 13-week study, all males and one female in the 120 mg/kg groups and 2 of 10 male rats in the 60 mg/kg group died during the first week of the study (Table 3). Clinical findings of toxicity in the 13-week study were dose dependent and included lethargy, lacrimation, tremors, convulsions, ataxia, and abnormal breathing. These effects were observed within minutes of dosing and disappeared within several hours after dosing.

BODY WEIGHT AND WEIGHT GAIN
Among groups scheduled for the 32-day interim evaluation, males in the 60 mg/kg bw group and the remaining male in the 120 mg/kg bw group had a significantly lower final mean body weight and mean body weight gain than those of the vehicle controls.

Among groups scheduled for evaluation at the end of the 13-week study, male rats in the 60 mg/kg group and females in the 120 mg/kg group had significantly lower final mean body weights and mean body weight gains than those of the vehicle controls (Table 3).

HAEMATOLOGY
On day 32, a minimal anaemia, evidenced by decreased hematocrit values, haemoglobin concentrations, and erythrocyte counts, was observed in males administered 60 or 120 mg/kg and females administered 30 mg/kg or greater.

By week 13, the anaemia had ameliorated and was evidenced only by minimal decreases in haemoglobin concentrations in males in the 60 mg/kg group and females in the 120 mg/kg group. On day 32, there was an indication of an erythropoietic response to the anaemia, evidenced by minimally increased reticulocyte counts in females administered 60 or 120 mg/kg. This was consistent with the minimal severity of the anaemia resulting in a weak stimulus for an erythropoietic response. Other changes in haematology variables were minor and sporadic.

CLINICAL CHEMISTRY
Dose-related, significant increases in cyanide and thiocyanate concentrations occurred in males and females at all time points; this would be consistent with the release of cyanide in vivo through the metabolism of methacrylonitrile via the cytochrome P450 enzymes.

On day 4, dose-related decreases in bile salt concentrations occurred in males and females; this suggests a decrease in the reabsorption of bile acids from the intestinal tract or altered synthesis of bile acids in the liver.

On days 4 and 32, serum alanine aminotransferase activities were significantly decreased in males in the 60 mg/kg group and in all dosed groups of females. This could represent an alteration in enzyme synthesis, release, catabolism, or inhibition by methacrylonitrile or its metabolites. The mechanism for the decreased enzyme activities is unknown. At week 13, there was evidence of increased hepatocellular leakage and/or altered function in dosed males, as demonstrated by increased serum sorbitol dehydrogenase and alanine aminotransferase activities and bile salt concentrations.
Also at 13 weeks, minimal increases in urea nitrogen concentrations occurred in all dosed groups of males and all but the 7.5 mg/kg group of females. Urea nitrogen concentrations were also generally increased on days 4 and 32 in males administered 15, 30, or 60 mg/kg and on day 32 in females administered 60 or 120 mg/kg; the mechanism was unknown, but the increases were minimal and were not considered clinically relevant.

ORGAN WEIGHTS
At the end of the 13-week study, liver weights of males that received 30 or 60 mg/kg were greater than those of the vehicle controls (Table 5). The absolute stomach weight of males in the 60 mg/kg group and the relative stomach weights of all dosed groups of males were significantly greater than those of the vehicle controls, as were the absolute and relative stomach weights of females that received 60 or 120 mg/kg. Absolute thymus weights of females that received 60 or 120 mg/kg and the relative thymus weight of the 120 mg/kg group were less than those of the vehicle controls. The relative lung weights of males in the 30 and 60 mg/kg groups and the relative heart, right kidney, and liver weights of females in the 120 mg/kg group were significantly greater than those of the vehicle controls. Relative heart, kidney, and liver weights were significantly increased in female rats that received 120 mg/kg.

GROSS PATHOLOGY
At necropsy, no gross lesions were attributed to methacrylonitrile administration.

HISTOPATHOLOGY:
Microscopically, the olfactory epithelium of the nasal cavity was identified as the primary target of methacrylonitrile toxicity in rats (Table 6). Treatment-related changes in the olfactory mucosa were limited to male and female rats in the 60 and 120 mg/kg groups and consisted of necrotic and metaplastic effects. Olfactory epithelial necrosis was characterized by the presence of cells undergoing various stages of necrosis, which resulted in epithelial erosion and the presence of necrotic cellular debris within the nasal passages. Metaplasia was characterized by the replacement of the injured olfactory epithelium with respiratory epithelium and/or an undifferentiated type of epithelium. The undifferentiated type of epithelium was characterized by attenuated cells covering denuded areas. Olfactory necrosis and metaplasia were usually unilateral lesions. Olfactory toxicity was more apparent in females than in males due to the higher survival rate of females in the 120 mg/kg group; in females, dose-related increases in the incidence and severity of olfactory lesions occurred at the 32-day interim evaluation and at 13 weeks. A time-dependent progression in the severity of olfactory lesions was not apparent. No microscopic effects were observed to account for organ weight differences.

OTHER FINDINGS
There were no significant differences in reproductive organ weights or sperm motility parameters between dosed and vehicle control males. Female rats that received 60 or 120 mg/kg had significantly longer estrous cycles than did the vehicle controls. Females in the 60 mg/kg group spent more time in diestrus than did the vehicle controls. The results are shown in Table 7.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
< 7.5 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
other: As significant increase in serum cyanide and thiocyanate as well as impaired clinical chemistry findings were observed in all treatment groups.
Dose descriptor:
NOAEL
Effect level:
7.5 mg/kg bw/day (nominal)
Sex:
female
Basis for effect level:
other: As impaired clinical chemistry findings were observed at 15 mg/kg bw and above.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 3: Survival and body weights of rats in the 13-week gavage study of methacrylonitrile

Dose [mg/kg bw]

Survivala

Mean body weightb[g]

Final weight relative to controls [%]

Initial

Final

Change

Males

0

10/10

121±2

326±4

205±3

 

7.5

10/10

123±1

320±2

198±2

98

15

10/10

116±2

318±3

202±2

97

30

10/10

120±2

321±7

202±7

99

60

8/10c

120±2

295±6**

175±5**

90

120

0/10c

120±2

-

-

-

Females

0

10/10

108±1

185±2

77±2

 

7.5

10/10

105±2

185±4

79±3

100

15

10/10

106±2

186±3

80±3

101

30

10/10

110±2

191±3

81±2

103

60

10/10

105±1

178±2

73±2

96

120

9/10c

109±1

171±3**

62±2**

93

** Significantly different (p≤0.01) from the vehicle control group by William’s test

aNumber of animals surviving at 13 weeks/number initially in group

bWeights and weight changes are given as mean ± standard error. Subsequent calculations are based on animals surviving to the end of the study

cWeek of death: 1

 

Table 4a: Selected clinical pathology data for male rats in the 13-week gavage study of methacrylonitrilea

 

Vehicle control

7.5 mg/kg

15 mg/kg

30 mg/kg

60 mg/kg

120 mg/kg

Haematology

n

10

10

10

9

8

0

Haematocrit [%]

46.6±0.3

45.4±0.4

47.5±0.7

45.9±0.4

45.3±0.4

-

Haemoglobin [µg/dL]

16.0±0.1

15.7±0.2

16.1±0.2

15.7±0.2

15.4±0.1 *

-

Erythrocytes [10^6 /µL]

8.57±0.08

8.56±0.06

8.87±0.13

8.52±0.11

8.43±0.07

-

Clinical chemistry

n

10

10

10

10

8

0

Cyanide [µmol/L]

0.37±0.05

0.62±0.08 **

1.03±0.07 **

1.70±0.13 **

1.82±0.17 **

-

Thiocyanate [µg/L]

158.4±6.3

429.6±8.7 **

596.4±16.2 **

735.1±23.7 **

790.1±9.2 **

-

Urea nitrogen [mg/dL]

21.1±0.5

23.5±0.4 **

24.8±0.5 **

25.1±0.5 **

24.0±0.3 **

-

Alanine aminotransferase [IU/L]

57±2

90±9 **

90±12 *

123±21 **

66±5

-

Sorbitol dehydrogenase [IU/L]

19±2

33±3 **

37±4 **

52±7 **

26±2 **

-

Bile salts [µmol/L]

18.9±2.7b

25.6±3.3c

22.7±1.3c

33.5±2.9 **c

26.9±4.4d

-

* Significantly different (p≤0.05) from the vehicle control group by Dunn’s or Shirley’s test

** p≤0.01

aData are given as mean ± standard error. Statistical tests were performed on unrounded data.

bn=5

cn=8

dn=6

 

Table 4b: Selected clinical pathology data for female rats in the 13-week gavage study of methacrylonitrilea

 

Vehicle control

7.5 mg/kg

15 mg/kg

30 mg/kg

60 mg/kg

120 mg/kg

Haematology

n

10

10

10

10

10

9

Haematocrit [%]

45.0±0.4

44.5±0.4

45.4±0.3

45.3±0.3

45.2±0.7

43.2±0.5

Haemoglobin [µg/dL]

15.2±0.2

15.3±0.1

15.5±0.1

15.4±0.2

15.3±0.2

14.6±0.2 *

Erythrocytes [10^6 /µL]

7.65±0.07

7.63±0.07

7.79±0.03

7.72±0.07

7.66±0.12

7.52±0.06

Clinical chemistry

n

10

10

10

10

10

9

Cyanide [µmol/L]

0.11±0.02

0.18±0.01 *

0.24±0.02 **

0.77±0.14 **

1.61±0.07 **

1.58±0.10 **

Thiocyanate [µg/L]

128.8±4.1

488.0±10.1 **

625.1±13.9 **

751.2±19.7 **

904.3±40.7 **

861.1±32.7 **

Urea nitrogen [mg/dL]

21.2±0.9

22.7±0.6

24.1±0.4 **

24.5±0.7 **

24.0±0.3 **

25.6±0.9 **

Alanine aminotransferase [IU/L]

52±2

84±12

78±10

56±9

45±4

35±1 **

Sorbitol dehydrogenase [IU/L]

23±2

34±5

30±3

28±3

25±2

22±1

Bile salts [µmol/L]

39.2±4.8

36.8±3.4

46.9±3.3

34.1±2.5

32.3±2.2

31.2±4.1

* Significantly different (p≤0.05) from the vehicle control group by Dunn’s or Shirley’s test

** p≤0.01

aData are given as mean ± standard error. Statistical tests were performed on unrounded data.

 

Table 5: Selected organ weights and organ-weight-to-body-weight ratios for rats in the 13-week gavage study of methacrylonitrile

 

Vehicle control

7.5 mg/kg

15 mg/kg

30 mg/kg

60 mg/kg

120 mg/kg

Male

n

10

10

10

10

8

0

Necropsy body weight

327 ± 4

321 ± 2

320 ± 3

321 ± 7

293 ± 5**

-

Liver

Absolute

Relative

 

10.936 ± 0.235

33.45 ± 0.60

 

11.267 ± 0.205

35.04 ± 0.50

 

11.278 ± 0.185

35.28 ± 0.38

 

12.525 ± 0.364**

39.04 ± 0.87**

 

11.764 ± 0.549*

40.09 ± 1.39**

 

-

-

Lung

Absolute

Relative

 

1.448 ± 0.038

4.43 ± 0.09

 

1.459 ± 0.037

4.54 ± 0.11

 

1.508 ± 0.050

4.71 ± 0.13

 

1.556 ± 0.041

4.86 ± 0.14*

 

1.433 ± 0.041

4.89 ± 0.09**

 

-

-

Stomach

Absolute

Relative

 

1.360 ± 0.038

4.16 ± 0.09

 

1.445 ± 0.020

4.50 ± 0.06*

 

1.411 ± 0.028

4.41 ± 0.07*

 

1.439 ± 0.020

4.50 ± 0.10**

 

1.595 ± 0.036**

5.45 ± 0.09**

 

-

-

Female

n

10

10

10

10

10

9

Necropsy body weight

185 ± 3

185 ± 3

188 ± 2

192 ± 4

175 ± 2*

172 ± 3**

Heart

Absolute

Relative

 

0.633 ± 0.010

3.42 ± 0.03

 

0.626 ± 0.021

3.39 ± 0.11

 

0.630 ± 0.012

3.35 ± 0.05

 

0.662 ± 0.028

3.44 ± 0.11

 

0.625 ± 0.014

3.57 ± 0.06

 

0.657 ± 0.012

3.83 ± 0.08**

R. kidney

Absolute

Relative

 

0.645 ± 0.012

3.49 ± 0.05

 

0.643 ± 0.010

3.49 ± 0.06

 

0.672 ± 0.013

3.57 ± 0.04

 

0.686 ± 0.017

3.57 ± 0.05

 

0.632 ± 0.011

3.61 ± 0.06

 

0.634 ± 0.010

3.69 ± 0.04*

Liver

Absolute

Relative

 

6.079 ± 0.156

32.89 ± 0.75

 

5.982 ± 0.126

32.43 ± 0.54

 

6.262 ± 0.162

33.22 ± 0.53

 

6.548 ± 0.157

34.06 ± 0.60

 

5.824 ± 0.201

33.25 ± 1.08

 

6.594 ± 0.206

38.34 ± 1.13**

Stomach

Absolute

Relative

 

1.063 ± 0.035

5.75 ± 0.17

 

1.080 ± 0.035

5.86 ± 0.21

 

1.105 ± 0.025

5.87 ± 0.11

 

1.151 ± 0.033

6.00 ± 0.19

 

1.171 ± 0.028*

6.70 ± 0.19**

 

1.316 ± 0.040**

7.67 ± 0.28**

Thymus

Absolute

Relative

 

0.233 ± 0.012

1.26 ± 0.06

 

0.238 ± 0.014

1.29 ± 0.08

 

0.234 ± 0.010

1.24 ± 0.05

 

0.240 ± 0.012

1.24 ± 0.05

 

0.193 ± 0.015*

1.10 ± 0.09

 

0.150 ± 0.005**

0.87 ± 0.03**

* Significantly different (p≤0.05) from the vehicle control group by William’s test

** p≤0.01

aOrgan weights (absolute) and body weights are given in grams; organ-weight-to-body-weight rations (relative weights) are given as mg organ weight/g body weight (mean ± standard error)

 

Table 6: Incidence and Severity of Selected Nonneoplastic Nasal Lesions in Rats in the 13-Week Gavage Study of Methacrylonitrile

 

Vehicle control

7.5 mg/kg

15 mg/kg

30 mg/kg

60 mg/kg

120 mg/kg

Male

32-day interim evaluation

Number examined microscopically

10

10

10

10

10

6

Metaplasia

0

0

0

0

4* (1.0)c

1 (1.0)

Necrosis

0

0

0

0

3* (1.0)

1 (2.0)

13-week study

Number examined microscopically

10

10

10

10

10

7

Metaplasia

0

1 (1.0)

0

0

6** (1.0)

1 (1.0)

Necrosis

0

0

0

0

2 (1.0)

1 (1.0)

Female

32-day interim evaluation

Number examined microscopically

10

10

10

10

10

10

Metaplasia

0

0

0

0

6** (1.0)

10** (1.9)

Necrosis

0

0

0

0

5** (1.2)

10** (1.7)

13-week study

Number examined microscopically

10

10

10

10

10

10

Metaplasia

0

0

0

0

9** (1.1)

9** (1.8)

Necrosis

0

0

0

0

1 (1.0)

3 (1.0)

 

Table 7: Summary of estrus cycle characterization for female rats in the in the 13-Week Gavage Study of Methacrylonitrile

 

Vehicle control

30 mg/kg

60 mg/kg

120 mg/kg

n

10

10

10

9

Necropsy body weight [g]

185 ± 3

192 ± 4

175 ± 2*

172 ± 3**

Estrous cycle length [days]

4.95 ± 0.05

5.20 ± 0.20

5.75 ± 0.29**

6.06 ± 0.41**c

Estrous stages [% of cycle]

 

 

 

 

Diestrus

32.5

35.8

43.3

41.7

Proestrus

15.8

17.5

15.8

14.8

Estrus

25.0

29.2

20.0

23.1

Metestrus

15.0

15.8

17.5

14.8

Uncertain diagnosis

11.7

1.7

3.3

5.6

* Significantly different (p≤0.05) from the vehicle control group by Shirley’s test

** p≤0.01

aNecropsy body weight and estrous cycle length data are presented as mean ± standard error.

bEvidence shows that females in the 60 mg/kg group differ significantly (Wilkinson’s Criterion, p≤0.05) from the vehicle control females in the relative length of time spent in the estrous stages. Dosed females spent more time in diestrus than vehicle control females.

cEstrous cycle was longer than 12 days or unclear in one of nine animals.

Applicant's summary and conclusion

Conclusions:
The results clearly revealed that male rats are more sensitive than females to methacrylonitrile treatment. In the rat study, 19 males and one female administered 120 mg/kg and two males administered 60 mg/kg died during the first week of the study. Males in the 60 mg/kg group at the 32-day interim evaluation and at 13 weeks and females in the 120 mg/kg group at 13 weeks had significantly lower final mean body weights and body weight gains than did the vehicle controls; the surviving male in the 120 mg/kg group also weighed less than the controls at the 32-day interim evaluation. Clinical findings of toxicity were dose dependent and included lethargy, lacrimation, tremors, convulsions, ataxia, and abnormal breathing. There was hematologic evidence indicating that administration of methacrylonitrile induced minimal, normocytic, normochromic anemia. At the 32-day interim evaluation, a minimal dose-related anemia was evidenced by decreases in hematocrit values, hemoglobin concentrations, and erythrocyte counts in male and female rats. The anemia ameliorated by week 13. Administration of methacrylonitrile resulted in dose-related increases in serum thiocyanate and blood cyanide concentrations of male and female rats. These changes were expected and would be consistent with the in vivo metabolism of methacrylonitrile to cyanide. Blood cyanide concentrations were generally higher in males than in females, which may explain the higher sensitivity of males to the lethal effect of methacrylonitrile. There was also biochemical evidence of increased hepatocellular leakage and/or altered function in dosed male rats, suggesting that the liver may be a target organ for toxic effects of methacrylonitrile. Minimal, but significant, decreases in absolute right kidney and thymus weights (32-day interim evaluation) and increases in liver and stomach weights (week 13) occurred in male rats that received 60 mg/kg compared to the vehicle controls. In female rats, stomach weights of the 60 and 120 mg/kg groups were significantly greater and thymus weights of the 120 mg/kg group were significantly less than those of the controls on day 32 and at week 13; liver weights were also significantly greater in females in the 120 mg/kg group than in the vehicle controls on day 32. Male and female rats administered 60 mg/kg and females administered 120 mg/kg had significantly greater incidences of metaplasia of the nasal olfactory epithelium on day 32 and at the end of the study than did the vehicle controls; incidences of olfactory epithelial necrosis were also significantly greater in females in the 60 and 120 mg/kg groups than in the vehicle controls on day 32. Incidence and/or severity increased with increasing dose in females; however, the mortality in male rats administered 120 mg/kg made it difficult to assess the dose-response relationship in males. The no-observed-adverse-effect level for the nasal cavity of rats was 30 mg/kg.