Registration Dossier

Diss Factsheets

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
1995
Deviations:
yes
Remarks:
This study exceeded the OECD 407 study design by increasing the number of animals to 8/sex/dose, including recovery animals (5/sex) in the control and high dose groups, and including a urinalysis prior to terminal sacrifice.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3-chlorophthalic anhydride
EC Number:
204-179-8
EC Name:
3-chlorophthalic anhydride
Cas Number:
117-21-5
Molecular formula:
C8H3ClO3
IUPAC Name:
4-chloro-1,3-dihydro-2-benzofuran-1,3-dione

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)IGS BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
Forty three male and 43 female Crl:CD(SD)IGS BR rats were received in good health from Charles River Laboratories, Inc., Raleigh, North Carolina. The animals were approximately 42 days old at receipt. The females were nulliparous and nonpregnant. All animals were quarantined for approximately one week, during which time they were weighed, examined by a veterinarian and evaluated for ecto- and endo-parasites, clinical signs and acclimation to husbandry. All animals were considered to be in good health and suitable for use in this study. There were 84 animals (42 males and 42 females) assigned to the study during the quarantine period. All animals were uniquely identified prior to initiation of the study by eartag. Animals were randomly assigned by sex to the different groups such that the body weights by sex of all the groups were homogenous at treatment initiation. The weight variation of the study animals at initiation did not exceed +/- 20% of the mean weight for each sex. The method and numbers for identification were documented in the study records. The animals were individually housed during the quarantine period and upon initiation of the treatment period in solid-bottom polycarbonate cages with stainless steel wire lids with chip cage litter. All animal rooms were on a 12 hour light cycle per day and were air-conditioned. Temperature and relative humidity (RH) were continuously monitored, controlled and recorded. Temperature and RH readings over the course of the study were 71.1-73.1°F and 36.3-65.4%, respectively. The basal diet and city tap water were provided ad libitum, except during the period of fasting prior to blood collection when food was withheld.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
Test article formulations were prepared at 2.0, 20.0 and 60.0 mg/ml of 3-CLPA in corn oil, and were administered at 5 ml/kg. The appropriate amount of 3-CLPA was weighed into a beaker, the corn oil was added and stirred to mix. Two 20-mL samples were collected as analytical and archived samples. The bulk sample was transferred to amber bottles for dosing and stored under refrigerated conditions.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to the study start, test formulations of 3 CLPA in corn oil at concentrations (2 and 200 mg/ml) encompassing the range of doses to be employed in this study were assayed for homogeneity and stability. Samples (20 mL each) for homogeneity determination were collected from the top, middle and bottom strata of the 2 and 200 mg/ml dosing formulations for homogeneity analysis and analyzed in triplicate. Aliquots of the formulation from each concentration were then transferred to two amber bottles to be used for storage stability studies. From each formulation, one of the amber bottles was placed in the refrigerator (~ 4°C) and protected from light. The samples were determined to be homogenous and stable for at least 35 days under refrigerated storage conditions after high performance liquid chromatography (HPLC) analysis. Suspensions of 3-CLPA were formulated independently for each concentration and stored refrigerated. Dose formulations were prepared once during the study and analysis of the concentration of test substance in the dosing suspensions was performed on all doses and the control prior to dosing using high performance liquid chromatography. The mean of the analyzed samples was within 10% of nominal (91.2, 93.5, and 94.2% for the low, medium, and high dose formulations, respectively), and the % RSD (relative standard deviation) for triplicate analyses did not exceed 1.6%. Analyses of samples for the mid dose (20 mg/ml) were repeated once due to percent of nominal of 87.5%. After repeating the analysis using archived analytical aliquots, the percent nominal was still ~87.5 %. Therefore, this dose was reformulated and analyzed within acceptable limits (93.5%).
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
8 animals per sex per dose, with an additional 5/sex at 0 and 300 mg/kg/day for a 14-day recovery period
Control animals:
yes, concurrent vehicle
Details on study design:
Doses were selected based on a 10-day range-finding study that employed doses of 0, 100, 300 and 1000 mg/kg/day, administered by oral gavage at 5 ml/kg for 10 consecutive days. There was substantial toxicity at 1000 mg/kg/day, expressed as clinical signs of respiratory distress, reductions in body weight, reductions in liver and paired kidney weights in the males (absolute but not relative to body weight), and morbidity and/or death in 1 male and 1 female. At 300 mg/kg/day, toxic effects included death in 1 male and sporadic clinical signs of respiratory distress in both males and females. Therefore, based on these observations, the doses chosen for this study were 0, 10, 100, and 300 mg/kg/day.

For the main study, 3-CLPA was administered orally by gavage at 0, 10, 100 or 300 mg/kg/day once daily for 28 consecutive days to Crl:CD(SD)IGS BR rats. The vehicle was corn oil and the dose volume was 5 mL/kg for all groups. Groups 1 and 4 each consisted of 13 animals/sex and Groups 2 and 3 each consisted of 8 animals/sex. Individual doses were based on the most recently recorded body weights. Following 28 consecutive days of dose administration, 8 rats/sex/group were euthanized (primary necropsy; study week 4); the remaining 5 rats/sex in the control and high dose groups were euthanized following a 14-day recovery period (recovery necropsy; study week 6).

Examinations

Observations and examinations performed and frequency:
Observations and examinations performed and frequency: All animals were observed twice daily for mortality. Clinical examinations were conducted and recorded daily. Individual body weights and food consumption were recorded weekly. Functional observational battery (FOB) (observations included: home cage; handling; open field; sensory; neuromuscular and physiological, including grip strength) data were recorded for all animals prior to the initiation of dose administration and weekly thereafter, including once during the recovery period. On Day 22 of exposure, all animals were assessed for auditory function and motor activity.

Prior to necropsy, 5 of the 8 animals/sex/group, excluding the recovery animals, were housed overnight in metabolism cages and urine was collected. The urine was analyzed for the presence of glucose, bilirubin, ketones, specific gravity, blood, pH, protein, urobilinogen, nitrite and leukocytes. Prior to necropsy all animals were fasted overnight. Hematology and clinical chemistry parameters (see lists below) were evaluated in all study animals at necropsy. Blood was collected at the time of necropsy via cardiac puncture.
Sacrifice and pathology:
A complete necropsy was conducted on all study animals. Animals were euthanized at the scheduled necropsies by CO2 asphyxiation. Select tissues and organs were collected and/or weighed and placed in 10% neutral-buffered formalin (except as noted) (see lists below). Histopathologic examination was performed on all tissues listed below from all animals in the control and 1000 mg/kg/day groups at the scheduled primary necropsy. Due to treatment-related effects observed in the high-dose male and female stomachs and mesenteric lymph nodes, these organs were also examined from all of the recovery animals (males and females), in addition to the low- and/or mid-dose study males and females
Statistics:
For body weights, organ weights, feed consumption, grip strength, auditory startle response and motor activity, treatment groups were compared to the concurrent control group using either parametric ANOVA or robust linear regression models. The homogeneity of variance assumption was examined via Levene’s Test. If Levene’s Test indicated lack of homogeneity of variance, robust regression methods were used to evaluate overall treatment group differences and, when a significant treatment effect was present, to compare each exposed group to controls. If Levene’s Test did not reject the hypothesis of homogeneous variances, a standard ANOVA was used to evaluate the overall effect of treatment and if necessary, each exposed group was compared to the control via 2-tailed Dunnett’s Test. Body weights during the recovery period and organ weights from the recovery animals were analyzed using the Student’s t-test. A test for statistical outliers was performed on body weights, feed consumption and organ weights. If a value flagged as an outlier had no biologically sound reason for inclusion in the data, it was flagged as an outlier and excluded from summarization and analysis. If feed consumption data were designated as outliers, then the summarized data encompassing that study period also did not include that value.

Binary FOB outcomes were analyzed using Fisher’s Exact Test, followed by individual pairwise comparisons of exposed groups to control if the overall test was statistically significant. All tests were two tailed. Ordinal FOB outcomes were analyzed using exact two-sided p-values for standard nonparametric tests. This included exact p-values for the Kruskal-Wallis Test for overall heterogeneity among dose groups, followed by individual pairwise comparisons of exposed groups to control if the overall test was statistically significant. Grip strength and hindlimb foot splay were analyzed using either a parametric ANOVA or robust linear regression methods.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
MALES:
- Clinical observations exhibiting a clear dose-response pattern of incidence or severity included rooting (in the low-, mid-, and high-dose groups) and salivation (in the high-dose group only) prior to and postdosing, which were considered to be a behavioral response of taste aversion to the dosing formulations and not a toxic sign. Eleven of 13 males at 300 mg/kg/day exhibited audible breathing during one or more days of treatment.
- Audible breathing was still evidenced during the recovery period in 4 out of 5 males at 300 mg/kg/day.

FEMALES:
- A female in the high-dose recovery group was euthanized moribund on sd 29 due to an apparent dosing error. Clincal observations considered to be related to dosing included salivation predosing in the low-, mid-, and high-dose groups for 2, 1, and 4 animals, respectively. Rooting postdosing was seen in 13/13 high-dose females, but it was also seen in 7, 6, and 6 females in the control, low-, and mid-dose groups, respectively. Postdose rooting in the bedding and salivation are considered behavioral responses of taste aversion to the dosing formulations and not a toxic sign. The reason for the high incidence of rooting seen in the controls could not be determined.
- There were no treatment-related clinical observations
Mortality:
mortality observed, non-treatment-related
Description (incidence):
FEMALES:
- There were 2 unscheduled deaths for the high-dose recovery females during the dosing phase of the study.
- One recovery female was found dead on sd 10 and at necropsy had a blood clot in the chest and a misshapen spleen. The death was not attributed to treatment with 3-CLPA.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
MALES
- There were significant decreases in male body weight in the 10 and 300 mg/kg/day groups, but not for the 100 mg/kg/day group, compared to controls on sd 14, 21, and 27. Mean body weight at 300 mg/kg/day was 14, 18, and 21% lower than the control at sd 14, 21, and 27, respectively. There was a significant reduction in male body weight change at 300 mg/kg/day for sd 0-7, 7-14, 14-21, and for the entire dosing period (sd 0 27). In addition, body weight change was significantly reduced at 10 mg/kg/day for only the sd 7-14 period. Body weight changes for the 10 mg/kg/day group were considered to be unrelated to treatment due to lack of effect in the mid-dose group.
- At scheduled sacrifice, mean body weights were significantly lower (17%) at 300 mg/kg/day compared to controls. There were no significant differences in body weights compared to controls at 10 and 100 mg/kg/day.
- During the recovery period, there were significant reductions in body weight at 300 mg/kg/day compared to controls on sd 27, 35, and 42 (a 27, 24, and 22% decrease, respectively). Body weight change in the recovery period at 300 mg/kg/day was similar to the controls.
- There was a statistically significant (22%) decrease in terminal body weight between the control and high-dose recovery males.

FEMALES
- There were no significant differences in female body weight across treatment groups on sd 0, 7, 14, 21, and 27. For body weight change, there was a significant increase for sd 0-7 only at 100 mg/kg/day. This difference was not considered related to treatment, based on the lack of dose response.
- At scheduled sacrifice, mean body weights were equivalent across dose groups.
- There were no statistically significant differences in terminal body weight.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
MALES:
- There was a significant decrease in male feed consumption (expressed as g/day) in the 300 mg/kg/day group compared to the control for sd 7-14, 14 21, 21-27, and for the entire dosing period. There was a significant decrease in feed consumption (expressed as g/kg/day), but only for sd 0-7. There was no effect of treatment on feed consumption expressed as g/day or g/kg/day at 10 or 100 mg/kg/day. A significant decrease in consumption in the 100 mg/kg/day group for study day 0-7 was not considered related to treatment due to the lack of dose response and lack of effects at other time periods.
- There was a significant decrease in feed consumption expressed as g/day, but a significant increase when expressed as g/kg/day for all recovery intervals at 300 mg/kg/day.

FEMALES:
- There was no effect of treatment on feed consumption when expressed as g/day for any treatment interval. There was a significant decrease in female feed consumption (expressed as g/kg/day) only at 100 mg/kg/day from sd 7-14 compared to controls. This difference was not considered related to treatment based on the lack of dose response.
- There were no significant differences in feed consumption, expressed as g/day or g/kg/day.
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
Description (incidence and severity):
MALES:
- No hematological parameters were affected by treatment.

FEMALES:
- For hematological parameters, hemoglobin and hematocrit were significantly reduced at 300 mg/kg/day. MCHC was significantly increased at 100 mg/kg/day but was unaffected at all other doses; this difference was considered unrelated to treatment, due to the lack of dose response and the small magnitude of the change.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
MALES:
- No clinical chemistry parameters were affected by treatment.

FEMALES:
- Clinical chemistry parameters not significantly affected by treatment included: blood BUN, creatinine, glucose, total protein, albumin, total cholesterol, AST, ALT, sodium, potassium, and chloride concentrations.
Urinalysis findings:
not specified
Description (incidence and severity):
MALES:
- No urinalysis parameters (pH or specific gravity) were significantly different for any dose group.

FEMALES:
- Urinalysis was unaffected except for the pH which was significantly reduced at 300 mg/kg/day.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
MALES:
- FOB data for all males were not significantly different across groups during the 4 weeks of dosing except for the following: there was a significant increase in the number of defecations per animal at 300 mg/kg/day in Weeks 2 and 3, the average approach response score was significantly reduced at 10 and 300 mg/kg/day in Week 3, and the average forelimb grip strength was significantly reduced in Week 4 at 100 and 300 mg/kg/day. On sd 22, there were no significant treatment-related effects on auditory startle. There was significantly reduced 10-minute activity during interval 4 at 10 and 300 mg/kg/day during motor activity testing. None of these differences were considered treatment-related, based on the lack of consistency across time periods and/or effects on related measurements. There were no treatment-related findings for motor activity for any other intervals at any dose.
- There were no differences between the control and high-dose groups in FOB data during the recovery period except for significantly decreased forelimb grip strength in the high-dose animals; this difference was considered unrelated to 3 CLPA toxicity, based on the lack of effect on this measurement during the dosing period.

FEMALES:
- There were no significant differences among groups in FOB data for all females during the quarantine period and in weeks 1, 2, 3, and 4. There were also no treatment-related effects on auditory startle responses or in motor activity which was tested on sd 22.
- There were no differences considered to be related to 3-CLPA exposure between the control group and the high-dose recovery group in FOB data during the recovery period.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
MALES:
- There was no effect of treatment at any dose for absolute organ weights. Relative paired adrenal gland and testes weights (% sacrifice weight) were significantly increased at 300 mg/kg/day, and paired adrenal gland weight, relative to brain weight, was significantly increased at 100 mg/kg/day.
- At 300 mg/kg/day, there were significant decreases in absolute brain, heart, liver, and paired kidney weights. Due to the lack of effect on these organ weights during the dosing phase, these differences were considered to be the result of the smaller group size and/or the result of the lower body weights. Paired testes and epididymides weights relative to sacrifice body weight were significantly increased, and relative (% of brain weight) paired adrenal weight was significantly increased at 300 mg/kg/day.

FEMALES:
- There was no significant difference in any absolute or relative organ weights (% sacrifice weight) when compared to controls, except the relative weight (% brain weight) for the uterus was significantly increased (68%) at 300 mg/kg/day. This difference was not considered to be related to treatment with 3-CLPA, due to the lack of difference in the absolute weight of the uterus.
- There were no statistically significant differences in absolute and relative organ weights between the control and high-dose recovery females.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were 13, 8, 8, and 13 study and recovery males and 13, 8, 8, and 11 study and recovery females at 0, 10, 100, and 300 mg/kg/day, respectively, that were evaluated at the scheduled necropsies.

MALES:
- In males, gross necropsy findings exhibited no treatment- or dose-related pattern of incidence or severity at scheduled sacrifice.
- There were no observations at necropsy considered to be related to the toxicity or recovery from exposure to 3 CLPA.

FEMALES:
- One recovery female was found dead on sd 10 and at necropsy had a blood clot in the chest and a misshapen spleen. The death was not attributed to treatment with 3-CLPA.
- At scheduled sacrifice, gross necropsy findings exhibited no treatment- or dose-related pattern of incidence or severity.
- There were no treatment-related observations at necropsy.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
MALES:
- Microscopic findings included epithelial hyperplasia of the forestomach in 7/8 males at 300 mg/kg/day and 1/8 males at 100 mg/kg/day. Erosion of the forestomach epithelium was seen in 1 male at 300 mg/kg/day. In addition, subacute inflammation of the glandular stomach was seen in the high dose (7/8). No lesions of the stomach were seen at 10 mg/kg/day. Lymphoid hyperplasia was seen in 2 males at 300 mg/kg/day but not at 10 or 100 mg/kg/day.
- Hyperplasia of the forestomach lining was seen in 4 males in the high-dose group, with reduced severity from that observed in the primary sacrifice; no inflammation of the glandular stomach was present nor were any mesenteric lymph node lesions noted. No other treatment-related microscopic changes were observed.

FEMALES:
- Histopathologic evaluations indicated hyperplasia and/or degeneration of the forestomach epithelium were present in 8 females at 300 mg/kg/day and 5 females at 100 mg/kg/day, but none at 10 mg/kg/day. Inflammation of the glandular stomach was also present in the high dose (8 females) and the mid dose (3 females), but not the low dose. Hyperplasia of the mesenteric lymph nodes was present in 1 female each at 100 and 300 mg/kg/day. No other treatment-related microscopic changes were observed.
- Four females at 300 mg/kg/day exhibited hyperplasia of the forestomach epithelium, with reduced severity from that observed at the primary necropsy, and 1 female exhibited inflammation of the glandular stomach. No mesenteric lymph node lesions were evident after the recovery period. No other microscopic changes were considered related to 3-CLPA exposure.
Histopathological findings: neoplastic:
not examined

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
food consumption and compound intake
histopathology: non-neoplastic
organ weights and organ / body weight ratios
other: Effects noted in the forestomach are considered to be not adverse
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
haematology
urinalysis

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
System:
other: body weight, food consumption, organ weight, haematological and urine parameters
Organ:
adrenal glands
blood
testes
Treatment related:
yes
Dose response relationship:
not specified

Any other information on results incl. tables

Summary and Statistical Analysis of Select Male Body Weight and Body Weight Changes

                    3-CLPA (mg/kg/day)

Group:

0

10

100

300

N

13

8

8

13

Body Weights (g)

  SD 14

     Mean

313.4

285.6*

291.0

270.2***

  SD 21

     Mean

358.5

317.0*

328.8

293.0***

  SD 27

     Mean

390.6

341.1*

353.7

308.7***

Body Weight Changes (g)

  SD 0 to 7

     Mean

57.9

52.0

49.0

47.9*

SD 7 to 14

     Mean

56.7

36.9*

45.4

26.7***

SD 14 to 21

     Mean

45.1

31.4

37.8

22.8*

SD 0 to 27

     Mean

191.9

144.5

157.1

113.1***

Mean +/- S.E.M. included in report. 

* = p is less than 0.05; Dunnett’s test

*** = p is less than 0.001; Dunnett’s test

S.E.M. = Standard error of the mean

SD = Study day

N = Number of animals

Summary and Statistical Analysis of Select Male Feed Consumption

                    3-CLPA (mg/kg/day)

Group:

0

10

100

300

N

13

8

8

13

Feed Consumption (g/day)

  SD 0 to 7

     Mean

24.6

23.2

17.4F

22.9

  SD 7 to 14

     Mean

24.6

21.6

22.7

19.4**

  SD 14 to 21

     Mean

24.7

20.9

22.5

19.4**

  SD 21 to 27

     Mean

23.7

20.3

23.0

18.9*

  SD 0 to 27

     Mean

24.4

21.5

21.3

20.2**

Feed Consumption (g/kg/day)

  SD 7 to 14

     Mean

86.3

80.2

84.3

74.8FF

Mean +/- S.E.M. included in report. 

* = p is less than 0.05; Dunnett’s test

** = p is less than 0.01; Dunnett’s test

F = p is less than 0.05; Individual t-test for pairwise comparisons to control in robust regression model

FF = p is less than 0.01; Individual t-test for pairwise comparisons to control in robust regression model

S.E.M. = Standard error of the mean

SD = Study day

N = Number of animals

Summary and Statistical Analysis of Select Male Functional Observational (FOB) Data

 

                    3-CLPA (mg/kg/day)

Group:

0

10

100

300

N

13

8

8

13

Week 2

Average Number of Defecations per Animal

     Mean

0.2

0.0

0.4

1.3

Week 3

Average Approach Response Score

     Mean

1.6

1.1

1.5

1.2

Percent with a Pupil Response

     Mean

46.15

50.00

0.00d

7.69

Average Number of Defecations per Animal

     Mean

0.0

0.0

0.3

1.0

Week 4

Average Forelimb Grip Strength (kg)

     Mean

0.810

0.794

0.615FF

0.590FFF

Mean +/- S.E.M. included in report. 

= p is less than 0.05; Exact Fishers’ Test for individual dose group versus controls.

= p is less than 0.05; Exact Kruskal-Wallis Test for individual dose group versus controls.

= p is less than 0.01; Exact Kruskal-Wallis Test for individual dose group versus controls.

FF = p is less than 0.01; Individual t-test for pairwise comparisons to control in robust regression model

FFF = p is less than 0.001; Individual t-test for pairwise comparisons to control in robust regression model

S.E.M. = Standard error of the mean

SD = Study day

N = Number of animals

Summary and Statistical Analysis of Select Male Auditory Startle and Motor Activity Data

                    3-CLPA (mg/kg/day)

Group:

0

10

100

300

N

13

8

8

13

Auditory Startle

Maximum Amplitude (arbitrary unit) (Block 1)

Mean

734.18

480.06

272.93**

488.22

Motor Activity

10-Minute Activity during Interval 4

     Mean

89.67

15.13*

61.88

17.08*

Mean +/- S.E.M. included in report. 

*  = p is less than 0.05; Dunnett’s Test.

**  = p is less than 0.01; Dunnett’s Test.

S.E.M. = Standard error of the mean

SD = Study day

N = Number of animals

Summary and Statistical Analysis of Select Male Relative Organ Weights

                    3-CLPA (mg/kg/day)

Group:

0

10

100

300

     N

8

8

8

8

Sacrifice Body Weight (g)

     Mean

350.41

318.42

329.66

291.17*

Relative Paired Adrenal Gland Weight (% of sacrifice weight)

     Mean

0.0154

0.0153

0.0187

0.0201**

  Relative Paired Testis Weight (% of sacrifice weight)

     Mean

0.9263

0.9445

0.9618

1.0918*

Relative Paired Adrenal Gland Weight (% of brain weight)

     Mean

2.6636

2.4743

3.1626F

2.9868

Mean +/- S.E.M. included in report. 

* = p is less than 0.05; Dunnett’s test

** = p is less than 0.01; Dunnett’s test

F = p is less than 0.05; Individual t-test for pairwise comparisons to control in robust regression model

S.E.M. = Standard error of the mean

SD = Study day

N = Number of animals

Summary and Statistical Analysis of Select Recovery Male Body Weights During the Recovery Period

3-CLPA (mg/kg/day)

Group:

0

300

   N

5

5

Body Weight (g)

SD 27

     Mean

413.4

301.9DD

SD 35

     Mean

455.8

347.9DD

SD 42

     Mean

491.4

384.5D

Mean +/- S.E.M. included in report. 

D = p is less than 0.05; Student’s t-Test

DD = p is less than 0.01; Student’s t-Test

S.E.M. = Standard error of the mean

SD = Study day

N = Number of animals

Summary and Statistical Analysis of Select Recovery Male Organ Weights and Relative Organ Weights

3-CLPA (mg/kg/day)

Group:

0

300

   N

5

5

Sacrifice Body Weight (g)

     Mean

485.17

378.41D

Brain Weight (g)

     Mean

2.1389

1.8561D

Heart Weight (g)

     Mean

1.6188

1.2059D

Liver Weight (g)

     Mean

23.8398

17.2724D

Paired Kidney Weight (g)

     Mean

3.8593

3.1414D

Relative Paired Testis Weight (% of sacrifice weight)

     Mean

0.6891

0.8483DD

Relative Paired Epididymis Weight (% of sacrifice weight)

     Mean

0.2412

0.2894DD

Relative Paired Adrenal Gland Weight (% of brain weight)

     Mean

2.3743

3.0330D

Mean +/- S.E.M. included in report. 

D = p is less than 0.05; Student’s t-Test

DD = p is less than 0.01; Student’s t-Test

S.E.M. = Standard error of the mean

SD = Study day

N = Number of animals

Summary and Statistical Analysis of Select Female Body Weight and Body Weight Changes

                    3-CLPA (mg/kg/day)

Group:

0

10

100

300

N

13

8

8

13

Body Weight Changes (g)

SD 0 to 7

     Mean

20.8

17.9

24.7FF

23.1

Mean +/- S.E.M. included in report. 

FF = p is less than 0.01; Individual t-test for pairwise comparisons to control in robust regression model

S.E.M. = Standard error of the mean

SD = Study day

N = Number of animals

Summary and Statistical Analysis of Select Female Feed Consumption Data

                    3-CLPA (mg/kg/day)

Group:

0

10

100

300

     N

13

8

8

13

Feed Consumption (g/kg/day)

  SD 7 to 14

     Mean

75.3

76.7

67.0*

75.3

Mean +/- S.E.M. included in report. 

* = p is less than 0.05; Dunnett’s test

S.E.M. = Standard error of the mean

SD = Study day

N = Number of animals

Summary and Statistical Analysis of Select Female Relative Organ Weights, Hematology and Urinalysis

                    3-CLPA (mg/kg/day)

Group:

0

10

100

300

     N

8

8

8

8

Sacrifice Body Weight (g)

     Mean

214.13

213.40

214.93

215.28

Uterus Weight (g)

     Mean

0.7259

0.7029

0.6573

1.0000

Relative Uterus Weight (% of brain weight)

     Mean

39.3290

37.9154

35.6166

58.1919*

Hematology

  Hemoglobin (g/dL)

     Mean

14.8

14.5

14.5

13.6**

  Hematocrit (%)

     Mean

37.5

35.9

35.7

34.5**

  Mean Corpuscular Hemoglobin Concentration (%)

     Mean

39.5

40.3

40.8*

39.5

Urinalysis

     N

5

5

5

5

  pH

     Mean

6.8

6.7

6.3

6.1**

Mean +/- S.E.M. included in report.

* = p is less than 0.05; Dunnett’s test

** = p is less than 0.01; Dunnett’s test

S.E.M. = Standard error of the mean

SD = Study day

N = Number of animals

Applicant's summary and conclusion

Conclusions:
The NOAEL was determined to be 100 mg/kg bw/day.
Executive summary:

The potential for repeated dose toxicity was determined in an OECD 407 study and in compliance with GLP criteria. In this study, 8 males and 8 females Crl:CD(SD)IGS BR per dose group were exposed to the test substance of 0, 10, 100 and 300 mg/kg bw/day for 28 consecutive days. The test doses, that were based on a 10-day range-finding study, were administered orally gavage. For the doses 0 and 300 mg/kg bw/day, an additional 5 animals per sex were included for a 14-day recover period. Observations included daily clinical examinations, the recording of body weight and food consumption, behavioral testing (FOB), urinalysis, clinical chemistry and haematology. Additionally, all animals were examined at necropsy and organ weights were determined. Treatment-related effects in males at 300 mg/kg/day were observed that included reductions in body weight, body weight change, and feed consumption. No treatment-related effects were observed in females at any dose during the in-life portion of the study. After the recovery period, body weights of male rats remained reduced, showing no indication of recovery. No indication of neurotoxic or immunotoxic effects were seen in this study. Microscopic lesions of the stomach, predominantly epithelial hyperplasia of the forestomach and/or inflammation of the glandular stomach, were seen in both sexes at 100 and 300 mg/kg/day and persisted in the high dose into the recovery period, although at reduced severity. No stomach lesions were present in either sex at 10 mg/kg/day. The effects on the (fore)stomach are not considered to be adverse. Based on these results, the NOAEL was determined to be 100 mg/kg bw/day.