4-chlorophthalic anhydride

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

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 groups, and including a urinalysis prior to terminal sacrifice.

GLP compliance:

yes

Remarks:

U.S. EPA TSCA GLP and OECD GLP

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-chlorophthalic anhydride

EC Number:

204-251-9

EC Name:

4-chlorophthalic anhydride

Cas Number:

118-45-6

Molecular formula:

C8-H3-Cl-O3

IUPAC Name:

5-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 assigned by sex to the different groups by means of stratified randomization 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 65.5-77.5°F and 35.6 66.2%, respectively. Minor deviations to the protocol-mandated ranges did not affect the design, conduct or conclusion of this study. The basal diet and city tap water was 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 4-CLPA in corn oil at 5 ml/kg. The appropriate amount of 4-CLPA was weighed into a beaker and a portion of the corn oil was added and stirred to mix. The remaining corn oil was added to the calibration mark. The formulations were then stirred. 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 4 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. Triplicate samples (1 mL each) for homogeneity determination were collected from the top, middle and bottom strata of the 2 and 200 mg/kg/day dosing formulations for homogeneity analysis. 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, and the other inside a metal can on a hood. The samples were determined to be homogenous and stable for at least 35 days under refrigerated or ambient storage conditions after high performance liquid chromatography (HPLC) analysis. Suspensions of 4-CLPA were formulated independently for each concentration and stored refrigerated. Dose formulations were prepared twice 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 (97, 102, and 101% in the first formulation for the low-, medium-, and high-dose formulations, respectively, and 99, 105, and 94% in the second formulation for the low, medium, and high-dose formulations, respectively), and the % RSD (relative standard deviation) for triplicate analyses did not exceed 1.5%. No 4-CLPA was detected in the vehicle control formulations, with an estimated limit of detection of 0.11 mg/ml.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

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 two 10-day range-finding studies that employed doses of 0, 100, 300 and 1000 mg/kg/day in the first study, and 0, 300, 500, and 700 in the second study, administered by oral gavage at 5 ml/kg for ten days. There was excess toxicity at 1000 mg/kg/day. At 500 and 700 mg/kg/day there was morbidity, mortality, weight loss, and clinical signs. At 300 mg/kg/day, there was weight loss and clinical signs of toxicity. Therefore, based on these observations, the doses chosen for this study were 0, 10, 100, and 300 mg/kg/day.

For the main study, 4-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:

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 19 of exposure, all animals were assessed for auditory function, motor activity and grip strength.

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. In addition, the stomachs and mesenteric lymph nodes from the animals in the low, mid, and recovery groups were examined.

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:
- Treatment-related clinical observations included rooting postdosing in one male at 10 mg/kg/day and all males at 100 and 300 mg/kg/day; salivation prior to dosing in nine males at 300 mg/kg/day and in one male at 100 mg/kg/day and salivation post dosing in one male at 300 mg/kg/day; sneezing in ten males at 300 mg/kg/day and one male at 100 mg/kg/day; and struggling during dosing in two males at 100 mg/kg/day and five males at 300 mg/kg/day. In 12 males at 300 mg/kg/day, and seven males at 100 mg/kg/day, audible respiration was reported in addition to one male at 100 mg/kg/day and two males at 300 mg/kg/day with labored breathing; two males at 100 mg/kg/day, and six males at 300 mg/kg/day with gasping following dosing and one male at 300 mg/kg/day gasping prior to dosing. Audible respiration appeared on sd 0, increased on sd 1 through sd 7, and occurred sporadically through sd 21.
- Recovery group: audible respiration persisted throughout the recovery period (sd 28 42) in one to three animals in the high-dose recovery group.

FEMALES:
- Treatment-related clinical observations included 13 females at 300 mg/kg/day and five females at 100 mg/kg/day with audible respiration; four females at 300 mg/kg/day and one female at 100 mg/kg/day gasping postdosing and two females at 300 mg/kg/day gasping prior to dosing; and two females at 300 mg/kg/day with labored breathing. Coughing in one female at 100 mg/kg/day; salivation prior to dosing in one female at 0 mg/kg/day, two females at 100 mg/kg/day, and ten females at 300 mg/kg/day; rooting (postdosing) in six, five, eight, and 13 females, respectively, at 0, 10, 100, and 300 mg/kg/day, probably due to taste aversion; sneezing in five and six females at 100 and 300 mg/kg/day, respectively; and struggling during dosing in three, one, two, and nine females, respectively, at 0, 10, 100, and 300 mg/kg/day were also considered related to treatment. Other clinical findings did not exhibit a clear dose-response pattern of incidence or severity.
- Recovery group: the only clinical observations in the recovery period included one female with alopecia at 0 mg/kg/day, one female each with audible respiration and rooting at 300 mg/kg/day, and one female with a curled tail at 300 mg/kg/day

Mortality:

mortality observed, non-treatment-related

Description (incidence):

MALES:
- No unscheduled deaths occurred in the study or recovery males in any dose group.

FEMALES:
- In the 300 mg/kg/day group, one female was found dead on sd 6 and one female was euthanized moribund on sd 19, both due to apparent misdirected dose.
- Recovery group: no unscheduled deaths occurred in the recovery females in any dose group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

MALES:
- There were significant decreases in male body weight in the 300 mg/kg/day group compared to controls on sd 7, 14, 21, and 28 (19, 18, 21, and 22%, respectively), and in the 100 mg/kg/day group compared to controls on sd 28 (10% decrease). There were no significant reductions in body weight on any study day in the 10 mg/kg/day group. There were significant reductions in male body weight change in the 300 mg/kg/day group compared to controls from sd 0 to 7 (83% decrease), sd 14 to 21 (47% decrease) and sd 0 to 28 (50% decrease). There was a significant decrease in body weight change at 100 mg/kg/day from sd 0 to 7 (22% decrease) and from sd 0 to 28 (24% decrease). There were no significant differences in body weight change in the 10 mg/kg/day group compared to controls.
- Body weights at the time of FOB in Weeks 1, 2, 3, and 4 were significantly reduced for males in the 300 mg/kg/day group compared to the controls. In addition, body weights for males in the 100 mg/kg/day group were significantly reduced compared to the control group in Week 4 at the time of FOB.
- At scheduled sacrifice, mean body weights were significantly (18%) lower in the 300 mg/kg/day group compared to controls.
- Recovery group: body weights measured on sd 35-42, during the recovery period, remained significantly decreased in the high-dose recovery group compared to the control group. However, body weight change was significantly increased in this group from sd 28 to 35, sd 35 to 42, and sd 28 to 42, compared to the control group, suggesting some tendency for recovery.
- Recovery group: there were no differences between the control and high-dose groups in FOB data during the recovery period, except for a significant decrease in body weight of the high-dose group compared to the controls.
- There was a statistically significant (21%) decrease in terminal body weight of the high-dose recovery males compared to the controls.

FEMALES:
- There were no significant differences in female body weight between the treated and control groups on sd 0, 14, 21, and 28. On sd 7, there was a 9% decrease in body weight at 300 mg/kg/day compared to controls. For body weight change, the sd 0 to 7 interval showed a significant (60.1%) reduction in the 300 mg/kg/day group when compared to the control group, and the sd 21 to 28 interval showed a significant 22.4% reduction in the 100 mg/kg/day group compared to controls.
- At scheduled sacrifice, mean body weights were equivalent across all groups.
- Recovery group: during the recovery period, there was a significant decrease in body weight in the 300 mg/kg/day group at sd 28 and 35 but no significant difference at sd 42 compared to controls. Body weight change was not significantly different between groups during the recovery period. .
- Recovery group: body weights obtained during recovery FOB evaluations were significantly reduced at 300 mg/kg/day compared to the control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

MALES:
- There were significant effects of treatment on male feed consumption (expressed as g/day and g/kg/day). From sd 0 to 7, there were significant (12% and 38%) reductions in feed consumption (expressed as g/day) in the 100 and 300 mg/kg/day groups, respectively. From sd 14 to 21, sd 21 to 27, and sd 0 to 27, there were significant decreases (19, 14, and 20%, respectively) in feed consumption (expressed as g/day) in the 300 mg/kg/day group compared to controls. From sd 0 to 7, there were significant (10% and 32%) reductions in feed consumption (expressed at g/kg/day), in the 100 and 300 mg/kg/day groups, respectively. From sd 7 to 14, there was a significant (9.26%) increase in feed consumption (expressed as g/kg/day) in the 300 mg/kg/day group compared to controls. Feed consumption (g/kg/day) was not calculated from sd 21 to 27 or sd 0 to 27 since the animals were not weighed on sd 27 (because they were fasted prior to necropsy).
- Recovery group: there were significant reductions in feed consumption (expressed as g/day) from sd 28 to 35, and significant increases in feed consumption (expressed as g/kg/day) from sd 28 to 35, sd 35 to 42, and sd 28 to 42 in the high-dose recovery group.

FEMALES:
- There were significant decreases in female feed consumption (expressed as g/day) in the 300 mg/kg/day group for sd 0 to 7 and sd 0 to 27 compared to controls, and significant decreases in feed consumption (expressed as g/kg/day) in the 300 mg/kg/day group from sd 0 to 7 and in the 100 mg/kg/day group from sd 7 to 14 compared to the controls. Feed consumption (g/kg/day) was not calculated for sd 21 to 27 or sd 0 to 27 since the animals were not weighed on sd 27, because they were fasted prior to necropsy.
- Recovery group: feed consumption (expressed as g/day) were not significantly different between groups during the recovery period.
- Recovery group: feed consumption (expressed as g/kg/day) was significantly greater in the 300 mg/kg/day group versus controls from sd 28 to 35, sd 35 to 42, and sd 28 to 42 (the entire recovery period).

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:
- For hematological parameters, MCV and MCH were significantly reduced in the 300 mg/kg/day group compared to controls.

FEMALES:
- WBC, nucleated RBC, corrected WBC and RBC counts, HGB, HCT, MCV, MCH, MCHC and % RBC distribution width, PLT count, MPV, segmented neutrophils, lymphocytes, monocytes, eosinophils, and PT were not affected by treatment.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

MALES:
- The blood clinical chemistry parameters affected by treatment included significant decreases in total protein and albumin in the 300 mg/kg/day group compared to the control group.

FEMALES:
- Blood urea nitrogen, creatinine, glucose, total cholesterol, AST, ALT, sodium, potassium, and chloride were not affected by treatment.
- There were significant decreases in total protein and albumin at 100 and 300 mg/kg/day.

Urinalysis findings:

no effects observed

Description (incidence and severity):

MALES:
- Urinalysis parameters were unaffected by treatment.

FEMALES:
- No urinalysis parameters were significantly different for any dose group.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

MALES:
- An increase in the incidence of altered gait was observed in males from the high-dose group in Week 4. No other treatment-related differences in FOB data, including grip strength, during the four weeks of the dosing period were observed. Occasional statistical differences were not consistent across weeks, and/or there was no pattern of related responses within the session. There were no significant treatment-related effects on motor activity or auditory startle evaluations performed on sd 19, or on grip strength that was conducted in week 4.
- Recovery group: there were no differences between the control and high-dose groups in FOB data during the recovery period

FEMALES:
- There were no significant treatment-related effects on auditory startle responses measured over five consecutive ten-trial blocks on sd 19. There were no statistically significant treatment-related effects on grip strength during Week 4, or on total motor activity on sd 19. There were significant decreases in motor activity in intervals 2 and 3 out of six total consecutive ten-minute intervals, in the 300 mg/kg/day group compared to controls. Since these differences were not consistent across time blocks, and the total motor activity counts were not significantly different across groups, they were not considered treatment-related.
- Recovery group: FOB data were not significantly different between groups in the recovery period.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

MALES:
- There were no significant differences in body weights or organ weights (absolute and relative to body weight and brain weight) at sacrifice compared to controls at 10 and 100 mg/kg/day. Absolute weights of the brain, spleen, paired testes, and paired epididymides were equivalent across all groups. Absolute thymus, heart, liver, and paired kidney weights were significantly lower in the 300 mg/kg/day group compared to controls, absolute paired adrenal gland weights were significantly greater in the 300 mg/kg/day group compared to controls, and the weight of the paired adrenal glands, relative to sacrifice body weight, also was significantly greater than the control value. In addition, the brain, testis, and epididymis weights, relative to body weight, were significantly greater in the 300 mg/kg/day group compared to the control group. Relative to brain weight, the thymus, heart, liver, and paired kidney weights of male rats in the 300 mg/kg/day group were all significantly less than the control values, and the paired adrenal gland weights were significantly greater in the 300 mg/kg/day group compared to controls. Spleen, paired testis, and paired epididymis weights relative to brain weight were unaffected across all groups.
- Recovery group: there were significant decreases in absolute brain, thymus, liver, spleen, and paired kidney weights compared to control weights, and significant increases in brain, paired testis, and paired epididymis weights relative to terminal sacrifice weight in the high-dose group compared to controls. There were no significant differences between groups for organ weights relative to brain weight.

FEMALES:
- Absolute weights of the brain, thymus, heart, liver, spleen, paired kidneys, paired adrenal glands, and uterus were equivalent across all groups. There was a significant (21%) increase in paired adrenal gland weights relative to terminal sacrifice weight in the 300 mg/kg/day group compared to controls. The weights of the brain, thymus, heart, liver, spleen, paired kidneys, and uterus relative to terminal body weights were unaffected across all groups. There were no significant differences in organ weights relative to brain weight; however, the thymus weight relative to brain weight was decreased (24%) at 300 mg/kg/day compared to controls.
- Recovery group: there were no statistically significant differences in absolute organ weights and organ weights relative to sacrifice weight or brain weight.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

MALES:
- Gross necropsy findings exhibited no treatment- or dose-related pattern of incidence or severity at scheduled sacrifice (these included alopecia in one male at 0 mg/kg/day, one male with a kidney abnormality at 300 mg/kg/day, and one male each with a sore on the head and neck at 0 mg/kg/day).
- Recovery group: the only gross necropsy findings were at 0 mg/kg/day (one male with alopecia and one with dark areas on the lungs). Since lesions were observed in the stomachs and mesenteric lymph nodes of the study males in the 300 mg/kg/day dose group, these tissues were evaluated in the recovery males.

FEMALES:
- Gross necropsy findings included two females at 100 mg/kg/day and three females at 300 mg/kg/day with thickened stomach linings. One female at 100 mg/kg/day had a fluid-filled uterus, most likely due to estrus at demise.
- Recovery group: gross findings at scheduled necropsy included one female at 300 mg/kg/day with hydrocephaly and one female at 0 mg/kg/day with a flattened portion of the right kidney. Also lesions were observed in the stomachs and mesenteric lymph nodes of the study females in the 300 mg/kg/day dose group.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

MALES:
- Histopathologic evaluations indicated a dose-related increase in incidence and severity of moderate hyperplasia of the forestomach and inflammation of the glandular stomach, and reactive lymphoid hyperplasia of the mesenteric lymph nodes at 300 mg/kg/day. Epithelial erosion and/or ulcers were observed in the forestomach of one male each from the 100 and 300 mg/kg/day groups. Because of the findings in the stomachs and lymph tissue from the high-dose group, stomachs and lymph tissue from animals in the mid-dose and high-dose groups were evaluated histopathologically. At 100 mg/kg/day, hyperplasia of the forestomach and mesenteric lymph node and inflammation of the glandular stomach were observed. Since lesions of the forestomach and mesenteric lymph nodes were observed at the mid dose, these tissues were examined histologically in the low-dose group. No lesions were observed in the stomach of the animals in the 10 mg/kg/day group; however, one animal in this group exhibited hyperplasia of the mesenteric lymph node.
- Recovery group: in the high-dose recovery group, hyperplasia of the forestomach and inflammation of the glandular stomach were still observed but were less severe than in the study group at 300 mg/kg/day, and no effects on mesenteric lymph nodes were observed, suggesting a recovery from the effects at the high dose.

FEMALES:
- Histopathologic evaluations indicated a dose-related increase in incidence and severity of moderate hyperplasia of the forestomach and inflammation of the glandular stomach, and reactive lymphoid hyperplasia of the mesenteric lymph nodes at 300 mg/kg/day. Therefore, the stomachs and lymph tissue from the mid-dose group were examined. At 100 mg/kg/day, hyperplasia of the forestomach and inflammation of the glandular stomach were observed; the incidence of lymphoid hyperplasia of the mesenteric lymph nodes was similar to that of the control group (one female in each group). Since lesions of the stomach and mesenteric lymph nodes were observed in the mid-dose group, these tissues also were evaluated in the low-dose group. No lesions were observed in the stomach or mesenteric lymph nodes at 10 mg/kg/day.
- Recovery group: since lesions were observed in the stomachs and mesenteric lymph nodes of the study females in the 300 mg/kg/day dose group, these tissues were evaluated in the recovery females. Hyperplasia of the forestomach (five females) and inflammation of the glandular stomach (one female) were observed in the 300 mg/kg/day recovery females, but were less severe than in study females at 300 mg/kg/day, and one female in the 300 mg/kg/day dose group had lymphoid hyperplasia of the mesenteric lymph nodes. The reduced severity of the stomach lesions suggests partial recovery.

Histopathological findings: neoplastic:

not examined

Details on results:

There were 8, 8, 8, and 8 study males and 8, 8, 8, and 6 study females at 0, 10, 100, and 300 mg/kg/day, respectively, that were evaluated at the scheduled necropsy. In addition, there were five recovery males and five recovery females each in the 0 and 300 mg/kg/day groups that were evaluated at scheduled necropsy.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

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

	4-CLPA (mg/kg/day)
Group:	0	10	100	300
N	13	8	8	13
Body Weights (g)
SD 7
Mean	276.4	268.5	262.8	223.7***
SD 14
Mean	324.5	307.0	303.3	265.9***
SD 21
Mean	362.1	344.1	334.3	285.7***
SD 28
Mean	379.2	345.8	340.5*	295.3***
Body Weight Changes (g)
SD 0 to 7
Mean	61.4	56.5	47.8**	10.4***
SD 14 to 21
Mean	37.6	37.1	31.0	19.8**
SD 0 to 28
Mean	164.3	133.9	125.5*	82.1***
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 *** = 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

	4-CLPA (mg/kg/day)
Group:	0	10	100	300
N	8	8	8	8
Feed Consumption (g/day)
SD 0 to 7
Mean	23.9	24.3	21.1**	14.7***
SD 14 to 21
Mean	23.7	23.6	22.0	19.3**
SD 21 to 27
Mean	23.4	23.1	22.6	20.1*
SD 0 to 27
Mean	23.6	23.6	21.9	18.8*
Feed Consumption (g/kg/day)
SD 0 to 7
Mean	97.8	101.1	88.1**	66.6***
SD 7 to 14
Mean	78.8	81.6	78.5	86.1*
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 *** = p is less than 0.001; Dunnett’s test Feed Consumption (g/kg/day) for SD 21 to 27 and SD 0 to 27 were not calculated as body weights were not collected on SD 27. 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

	4-CLPA (mg/kg/day)
Group:	0	10	100	300
N	13	8	8	13
Week 1
Body Weight (g)
Mean	218.98	215.19	218.53	203.92*
Percent with Piloerection
Mean	0.00	0.00	12.50	38.46F
Week 2
Body Weight (g)
Mean	271.68	264.59	259.76	220.27***
Average Pupil Size Score
Mean	2.1	1.5	1.4	1.4
Percent with a Pupil Response
Mean	92.31	50.00F	37.50F	46.15F
Week 3
Body Weight (g)
Mean	319.45	307.12	303.23	264.36***
Week 4
Body Weight (g)
Mean	357.98	340.30	320.05d	283.85ddd
Percent with Abnormal Gait (open field observations)
Mean	0.00	0.00	12.50	61.54FF
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 = 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. F = p is less than 0.05; Exact Fishers’ Test for individual dose group versus controls. FF = p is less than 0.01; Exact Fishers’ Test for individual dose group versus controls. d  = p is less than 0.05;  Individual t-test for pairwise comparisons to control in robust regression model ddd  = 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 Relative Organ Weights, Clinical Chemistry, and Hematology Parameters

	4-CLPA (mg/kg/day)
Group:	0	10	100	300
N	8	8	8	8
Sacrifice Body Weight (g)
Mean	354.82	343.16	337.06	291.47***
Thymus Weight (g)
Mean	0.5573	0.5684	0.5446	0.4218*
Heart Weight (g)
Mean	1.2688	1.3339	1.1922	1.0357**
Liver Weight (g)
Mean	13.9119	13.2873	12.3081	10.5841**
Paired Kidney Weight (g)
Mean	3.0882	2.8431	2.8494	2.4549***
Paired Adrenal Gland Weight (g)
Mean	0.0546	0.0604	0.0599	0.0705**
Relative Brain Weight (% of sacrifice weight)
Mean	0.5575	0.5833	0.6005	0.6795***
Relative Paired Adrenal Gland Weight (% of sacrifice weight)
Mean	0.0154	0.0175	0.0180	0.0244***
Relative Paired Testis Weight (% of sacrifice weight)
Mean	0.8941	0.9085	0.9246	1.0501*
Relative Paired Epididymis Weight (% of sacrifice weight)
Mean	0.2656	0.2772	0.2784	0.3071**
Relative Thymus Weight (% of brain weight)
Mean	28.4632	28.5341	26.8743	21.4047**
Relative Heart Weight (% of brain weight)
Mean	64.7087	66.7205	59.3103	52.3909**
Relative Liver Weight (% of brain weight)
Mean	708.2666	665.4075	610.4353	535.1261FFF
Relative Paired Kidney Weight (% of brain weight)
Mean	157.2041	142.5640	141.6892	124.4539***
Relative Paired Adrenal Gland Weight (% of brain weight)
Mean	2.7805	3.0170	2.9874	3.5848**
Clinical Chemistry
Total Protein (g/dL)
Mean	7.0	6.9	6.9	6.4FF
Albumin (g/dL)
Mean	4.6	4.5	4.4	4.1FFF
Hematology Parameters
Mean Corpuscular Volume (u^3)
Mean	62.3	61.5	62.0	57.4***
Mean Corpuscular Hemoglobin (pg)
Mean	23.1	23.2	23.1	21.3*
Mean +/- Standard error of the mean included in report.  * = p is less than 0.05; Dunnett’s test ** = p is less than 0.01; Dunnett’s test *** = p is less than 0.001; Dunnett’s test 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 SD = Study day;  N = Number of animals

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

	4-CLPA (mg/kg/day)
Group:	0	300
N	5	5
Body Weight (g)
SD 28
Mean	410.5	292.9DD
SD 35
Mean	440.1	333.7DD
SD 42
Mean	471.2	376.4DD
Body Weight Change (g)
SD 28 to 35
Mean	29.6	40.8D
SD 35 to 42
Mean	31.1	42.8DD
SD 28 to 42
Mean	60.7	83.5DD
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 Feed Consumption During the Dosing and Recovery Periods

	4-CLPA (mg/kg/day)
Group:	0	300
N	5	5
(g/day)
SD 0 to 7
Mean	23.4	13.4DDD
SD 7 to 14
Mean	24.4	18.8DD
SD 14 to 21
Mean	24.2	16.5DD
SD 21 to 28
Mean	24.6	18.1D
SD 0 to 28
Mean	24.1	16.7DDD
SD 28 to 35
Mean	27.3	23.6D
(g/kg/day)
SD 0 to 7
Mean	94.5	62.3DDD
SD 0 to 28
Mean	74.8	66.7DD
SD 28 to 35
Mean	64.1	75.8DD
SD 35 to 42
Mean	61.1	74.3DDD
SD 28 to 42
Mean	62.5	75.0DD
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 DDD = p is less than 0.001; 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 Functional Observational Battery Data During the Recovery Period

	4-CLPA (mg/kg/day)
Group:	0	300
N	5	5
Body Weight (g)
Mean	433.82	326.32DDD
Mean +/- S.E.M. included in report.  DDD = p is less than 0.001; 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

	4-CLPA (mg/kg/day)
Group:	0	300
N	5	5
Sacrifice Body Weight (g)
Mean	464.26	368.69DD
Brain Weight (g)
Mean	2.1276	1.9396DD
Thymus Weight (g)
Mean	0.6774	0.5326D
Liver Weight (g)
Mean	21.7053	16.9422D
Spleen Weight (g)
Mean	0.8285	0.6587D
Paired Kidney Weight (g)
Mean	3.5956	30.895D
Relative Brain Weight (% of sacrifice weight)
Mean	0.4583	0.5301D
Relative Paired Testis Weight (% of sacrifice weight)
Mean	0.7417	0.8072D
Relative Paired Epididymis Weight (% of sacrifice weight)
Mean	0.2601	0.2958D
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

	4-CLPA (mg/kg/day)
Group:	0	10	100	300
N	13	8	8	13^
Body Weight (g)
SD 7
Mean	206.5	208.5	205.8	187.0*
Body Weight Changes (g)
SD 0 to 7
Mean	21.8	24.4	20.9	8.7***
SD 21 to 28
Mean	4.5	-2.0	-5.6F	4.1
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 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 ^ = Starting with SD 7, the N=12; and starting with SD 21, the N=11.

Summary and Statistical Analysis of Select Female Feed Consumption

	4-CLPA (mg/kg/day)
Group:	0	10	100	300
N	8	8	8	7^
(g/day)
SD 0 to 7
Mean	17.5	17.3	17.0	11.6***
SD 0 to 27
Mean	16.8	16.2	15.8	13.5*
(g/kg/day)
SD 0 to 7
Mean	90.4	87.9	87.0	64.1FFF
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 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 ^ = Starting after SD 7, the N=6. Feed Consumption (g/kg/day) for SD 21 to 27 and SD 0 to 27 were not calculated as body weights were not collected on SD 27.

See Overall remarks, attachments section below for additional tables.

Applicant's summary and conclusion

Conclusions:

The NOAEL was determined to be 10 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 two range-finding studies, 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. For males, toxicity was present at 300 mg/kg/day, expressed as increased incidence of clinical signs (primarily associated with aversion to the dosing and probable reaction to the irritant effects of the test substance appearing as respiratory distress); decreases in body weight, body weight change, and feed consumption, expressed as g/day and g/kg/day; abnormal gait reported during open field observations in the Week 4 FOB; decreases in MCV and MCH, and decreases in total protein and albumin. The changes in organ weights for males were considered to be related to the decreased body weight and not a direct toxic effect of 4-CLPA, which was supported by the lack of histopathological findings. In males, there were no effects on grip strength (forelimb or hindlimb), startle response, or motor activity during the study, and no treatment-related gross findings. Histopathologic evaluation indicated a dose-related increase in incidence and severity of inflammation of the glandular stomach, hyperplasia of the forestomach, and reactive lymphoid hyperplasia of the mesenteric lymph nodes in the 100 and 300 mg/kg/day groups. Ulceration and/or epithelial erosion were also recorded in the forestomach of one male each from the mid- and high-dose groups. One lymphoid hyperplasia of a mesenteric lymph node was recorded in the 10 mg/kg/day group, but this was considered to be consistent with normal occurrence of this type of lesion in this rat strain (supported by the finding in one female control animal). There were no treatment-related lesions present in the stomachs at 10 mg/kg/day.

 

Females exhibited toxic effects at 300 mg/kg/day, including significant decreases in body weight gain and feed consumption (g/day and g/kg/day) from sd 0 to 7; transient body weight decreases as measured prior to FOB, reduced feed consumption from sd 0 to 27 (g/day); and clinical signs of respiratory distress (considered to be related to the irritant properties of the test substance). No effects on FOB, grip strength, startle response, or total motor activity were observed. The changes in adrenal weights for females were considered to be related to the decreased body weight and not a direct toxic effect of 4 CLPA, which was supported by the lack of histopathological findings. Decreases in total protein and albumin, as well as grossly observed stomach lesions, were recorded at 300 mg/kg/day. At 100 mg/kg/day, there were significant decreases in total protein and albumin, clinical signs of respiratory distress, and significant but transient decreases in feed consumption (sd 7 to 14 g/kg/day). Histopathologic evaluation indicated a dose-related increase in incidence and severity of inflammation of the glandular stomach, hyperplasia of the forestomach, and reactive lymphoid hyperplasia of the mesenteric lymph nodes in the 300 mg/kg/day group. Inflammation of the glandular stomach and hyperplasia of the forestomach also were observed in the 100 mg/kg/day group. Ulceration was also recorded in the forestomach of one female from the mid-dose group. Lymph node hyperplasia was observed in one control and one 100 mg/kg/day animal. No treatment-related stomach or lymph node lesions were present at 10 mg/kg/day. 

 

Lymphoid hyperplasia in the mid- and high-dose groups was considered to represent a reactive change due to the nature and severity of the stomach lesions and not from any direct treatment exposure. Although the pathogenesis of the stomach lesions could not be determined conclusively, the lesions appeared related to the high local exposure to the gavage-administered compound.

 

Body weight gain in males was increased compared to controls during the two-week recovery period, but the mean body weight for the high-dose recovery group remained decreased from controls throughout the period. In both males and females, after a two-week recovery period, no lesions of the mesenteric lymph nodes and minimal lesions of the glandular stomach were observed, while hyperplasia of the forestomach was still observed but at a lesser severity, thus indicating a significant recovery from the high-dose effects. 

 

In conclusion, gavage administration of 4-CLPA once daily at 0, 10, 100, or 300 mg/kg/day to young adult CD (SD) rats, for 28 consecutive days, resulted in a NOAEL of 10 mg/kg/day.