Benzeneacetyl chloride, 2,5-dimethyl-

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2006 to Jan 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

July 1995

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

470-780-9

EC Name:

-

Cas Number:

55312-97-5

Molecular formula:

C10 H11 Cl O

IUPAC Name:

2-(2,5-dimethylphenyl)acetyl chloride

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: R. Janvier, Le Genest St Isle, France.
- Age at study initiation: 6 weeks
- Weight at study initiation: 197 to 226 g for the males and 154 to 180 g for the females
- Housing: Rats were housed individually in suspended, stainless steel, wire-mesh cages.
- Diet (e.g. ad libitum): Certified rodent powdered and irradiated diet A04CP1-10 from S.A.F.E. (Scientific Animal Food and Engineering, Augy, France), ad libitum
- Water (e.g. ad libitum): filtered and softened tap water from the municipal water supply, ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY: Routine analyses of food and water indicated that there was no contamination which could have compromised the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light):12/12

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was incorporated into the diet by dry mixing to provide the required dietary concentrations. There was one preparation for each concentration. When not in use, the diet formulations were stored at approximately -18° C.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The homogeneity of test substance in diet was verified on the first formulation for the lowest and highest concentrations to demonstrate adequate formulation procedures. Dietary levels of the test substance were verified for each concentration. The stability of the dietary formulation was determined for a time period which covered the period of storage and usage for the current study (concentrations of 50, 100 and 2500 ppm). They were analyzed after having been frozen for up to 158 days followed by 10 days of storage at room temperature.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 500 ppm

Doses / concentrations 2

Dose / conc.:

500 ppm

Doses / concentrations 3

Dose / conc.:

100 ppm

No. of animals per sex per dose:

20

Control animals:

yes, plain diet

Details on study design:

- Fasting period before blood sampling for clinical biochemistry: over night

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily, detailed physical examinations were performed at least weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed twice during the acclimatization period, on the first day of test
substance administration, then at weekly intervals throughout the treatment period and before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: During the acclimatization period all animals were subjected to an ophthalmic examination.
After instillation of an atropinic agent (Mydriaticum, Merck Sharp and Dohme) each eye was examined by means of an indirect ophthalmoscope. During Week 4, animals from control and high dose groups were re-examined.
- Dose groups that were examined: control and high dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On study Days 30 or 31, blood samples were taken from all animals in all groups by
puncture of the retro-orbital venous plexus prior to sacrifice. An approximately equal number of animals randomly distributed amongst all groups were sampled on each day.
- Anaesthetic used for blood collection: Yes (identity) Isoflurane
- Animals fasted: Yes, over night
- How many animals: all

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see haematology
- Animals fasted: Yes
- How many animals: all


PLASMA/SERUM HORMONES/LIPIDS: Yes / No / Not specified
- Time of blood sample collection:
- Animals fasted: Yes / No / Not specified
- How many animals:

URINALYSIS: Yes
- Time schedule for collection of urine: On study Day 24, in the morning, overnight urine samples were collected from all animals in all groups.
- Metabolism cages used for collection of urine: Yes / No / Not specified
- Animals fasted: Yes, during urine collection

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During study Week 4, a neurotoxicity assessment was performed for all animals by observers
who were blind with respect to the dose level.
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY:No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs were observed throughout the course of the study.

Mortality:

no mortality observed

Description (incidence):

There were no mortalities during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 2500 ppm, mean body weight and mean body weight gain parameters in male animals were unaffected by treatment. In females, mean body weight was reduced by 6 to 7% between Study Days 15 and 29, when compared to the control values (not statistically significant). Between study Days 8 and 15, there was a mean body weight gain of +1.7g/day (statistically significant, p<0.05) in females, compared with a gain of +3.4g/day in the corresponding controls. Thereafter, mean body weight gain per day was comparable to controls in females. Overall between Study Days 1 and 29, the cumulated body weight gain was reduced by 25% in females (not statistically significant) when compared to the corresponding control values.
At 500 and 100 ppm, no effect on mean body weight or mean body weight gain parameters was observed in either sex.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 2500 ppm, mean food consumption in male animals was unaffected by treatment. Mean food consumption in females was decreased by between 8 and 15% each measured interval from Study Day 11 onwards, the effects being statistically significant (p<0.05) between study Days 11 to 15 and Study Days 22 and 25.
At 500 and 100 ppm, the mean food consumption in both sexes was unaffected by treatment.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related ophthalmological findings during the study in either sex.

Haematological findings:

no effects observed

Description (incidence and severity):

No treatment-related change was noted for the parameters assayed at any dietary level tested in either sex.
Slightly lower mean red blood cell count (-7%, not statistically significant), hemoglobin concentration (-8%, p<0.05) and hematocrit (-8%, p<0.05) were observed at 2500 ppm in females only. However in view of the variation of the individual values these differences were considered to be incidental.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Slightly lower mean total bilirubin concentration was noted at 2500 ppm in females (-50%, p<0.05).
No other treatment-related change was noted for the parameters assayed. The statistically significant difference observed in total bilirubin at 500 ppm in males was considered incidental in the absence of a similar change at 2500 ppm.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No treatment-related change was noted for the parameters assayed at any dietary level tested in either sex.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Exploratory locomotor activity
At 2500, 500 and 100 ppm in both sexes, no treatment-related changes were recorded in overall mean exploratory locomotor activity. In addition, the pattern of the locomotor activity over time was similar to controls. The slight decrease in overall mean exploratory locomotor activity noted at 2500 ppm in males (-27%, not statistically significant) was considered to be fortuitous and due to a particularly high mean value in the control group. Furthermore, the mean value observed in the high dose males for this parameter was within the in-house historical control range.
Open field observation
No treatment-related changes were recorded during the open field observation at any dose level in either sex. The only change noted in gait score was observed in one female at 2500 ppm in isolation and was thus considered not to be treatment-related.
Sensory reactivity
All reflexes and responses evaluated were unaffected by the treatment at any dose level in either sex. The few changes noted in both sexes for the flexor reflex and/or tail pinch response were observed in isolation and/or with no dose-relationship and were thus considered not to be treatment-related.
Grip strength
The fore- and hindlimb grip strength were unaffected by the treatment at any dose level in either sex. A slight decrease in hindlimb grip strength was observed in isolation in high dose males and females in comparison to controls (-19% and -17%, respectively, not statistically significant), but it was considered to reflect inter-individual variations and not to be a treatment-
related effect.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At 2500 ppm, a lower mean terminal body weight was observed in females when compared to controls (-6%, not statistically significant).
At 2500 ppm in females, mean absolute and relative liver weights were statistically significantly higher when compared to controls. These changes were considered to be treatment-related but not adverse. All other organ weight differences were judged to be incidental in view of their individual variation.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All the macroscopic changes were considered as incidental and not treatment-related.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In the thyroid gland, minimal follicular cell hypertrophy was observed in 1/5 males at 2500 ppm. In isolation, this finding was considered to be incidental and not treatment-related. All the other microscopic changes observed were considered as incidental and not treatment-related.

Effect levels

1

Key result

Dose descriptor:

NOEL

Effect level:

43.8 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

haematology

organ weights and organ / body weight ratios

2

Key result

Dose descriptor:

NOEL

Effect level:

194 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

2500 ppm in diet

Sex:

male

Basis for effect level:

other:

Remarks on result:

other: No effects observed up to the highest dose tested

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Dietary administration for 28 days to male and female Wistar rat Rj:WI (TOPS HAN) at dose levels of 100, 500 or 2500 ppm induced no mortality, no treatment-related clinical signs, no treatment-related changes in the exploratory locomotor activity, open field observation, sensory reactivity and grip strength, no ophthalmological findings, no changes in hematology or urinalysis parameters in both sexes. Up to the highest dose level of 2500 ppm, males were unaffected by the test item. Treatment-related findings were confined to female rats from the 2500 ppm group and consisted of slightly lower body weight, body weight gain, food consumption and total bilirubin concentration, and slightly higher liver weight.
The No Observed Effect Level (NOEL) in the rat, following 28 days administration was found to be 2500 ppm in males and 500 ppm in females, equating approximately to 194 mg/kg body weight/day in males and 43.8 mg/kg body weight/day in females.

Executive summary:

In a subacute toxicity study according to OECD guideline 407 (July 1995) 2,5-Dimethylphenylacetic acid (100 % a.i.) was administered to 20 Wistar rats/sex/dose in the diet at dose levels of 0 (control), 100, 500 and 2500 ppm for 28 consecutive days.

No mortality occurred. No clinical signs and no changes were observed at the weekly detailed clinical observations. In females, mean body weight was reduced by 6 to 7% between Study Days 15 and 29 in the 2500 ppm group, when compared to the control values (not statistically significant). Between study Days 8 and 15, there was a mean body weight gain of +1.7g/day (statistically significant, p<0.05) in females, compared with a gain of +3.4g/day in the corresponding controls. Thereafter, mean body weight gain per day was comparable to controls in females. Overall between Study Days 1 and 29, the cumulated body weight gain was reduced by 25% in females (not statistically significant) when compared to the corresponding control values.

Mean food consumption in females (2500 ppm) was decreased by between 8 and 15% each measured interval from Study Day 11 onwards, the effects being statistically significant (p<0.05) between study Days 11 to 15 and Study Days 22 and 25.

No treatment related changes in haematology were found. Slightly lower mean total bilirubin concentration was noted at 2500 ppm in females (-50%, p<0.05).

No other treatment-related change was noted for the parameters assayed. The statistically significant difference observed in total bilirubin at 500 ppm in males was considered incidental in the absence of a similar change at 2500 ppm. No relevant changes were found during urinalysis. Some effect on behaviour were found but considered to be non-treatment related because they were within the in-house historical ranges.

At 2500 ppm, a lower mean terminal body weight was observed in females when compared to controls (-6%, not statistically significant). At 2500 ppm in females, mean absolute and relative liver weights were statistically significantly higher when compared to controls. These changes were considered to be treatment-related but not adverse. All other organ weight differences were judged to be incidental in view of their individual variation.

 

On the basis of the results obtained in this study, the dose level of 43.8 mg/kg bw/day (500 ppm) in females and 194 mg/kg bw/d (2500 ppm) in males was considered the NOEL (No Observed Effect Level).