Anisole

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 15 February, 2012 to 1 May, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study, well conducted.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

yes

Remarks:

: these deviations are considered not to have affected the validity of the study (see below for more details).

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Remarks:

2012-12-05

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Anisole

EC Number:

202-876-1

EC Name:

Anisole

Cas Number:

100-66-3

Molecular formula:

C7H8O

IUPAC Name:

anisole

Test material form:

other: liquid stored at ambient temperature

Details on test material:

- Name of test material (as cited in study report): Anisole (methoxybenzene, methyl phenyl ether)
- Physical state: clear, colourless liquid
- Stability under test conditions: yes
- Storage condition of test material: ambient temperature

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: obtained from a colony maintained under SPF-conditions by Harlan Laboratories
- Age at study initiation: 7 weeks.
- Weight at study initiation: mean body weight just before exposure on day 0 was 199 and 176 g for males and females of the range finding study, and 242 and 181 g for males and females of the main study, respectively.
- Housing: the animals were housed three (range finding study) or five (main study) rats to a cage. During exposure, the rats were housed individually in the exposure unit. Immediately after each exposure, the animals were returned to their home cages.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 45-65%. Occasionally however, the relative humidity was higer than 65% for short periods of time, due to cleaning activities in the animal room.
- Air changes (per hr): 10 per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

nose only

Vehicle:

clean air

Remarks on MMAD:

MMAD / GSD: Not applicable

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: the animals were exposed to the test atmosphere in nose-only inhalation units (a modification of the design of the chamber manufactured by ADG Developments Ltd.,Codicote, Hitchin, Herts, SG4 8UB, United Kingdom; see Figure 1) consisting of a cylindrical polypropylene or stainless steel column, surrounded by a transparent cylinder.
- Exposure chamber volume: 50 litres
- Method of holding animals in test chamber: the animals were secured in plastic animal holders (Battelle), positioned radially through the outer cylinder around the central column. Male and female rats of each group were placed in alternating order. The remaining ports were closed. Only the nose of the rats protruded into the interior of the column. The animal's body does not exactly fit in the animal holder which always results in some leakage from the high to the low pressure side. By securing a positive pressure in the central column and a slightly negative pressure in the outer cylinder which encloses the entire animal holder, dilution of test atmosphere by air leaking from the animal’s thorax to the nose was avoided. Animals were rotated weekly (main study) or daily (range finding study) with respect to their position in the column.
- Source and rate of air: the total air flow through the units was at least 1 litre/min for each rat
- System of generating particulates/aerosols: not applicable
- Temperature, humidity, pressure in air chamber: temperature and relative humidity were recorded hourly during exposure.Measured temperature during exposure varied between 20.0 and 22.9°C, i.e. within the range of 19-25°C required by OECD guideline 412. For the control, low, mid and high
concentration groups, mean temperature (± standard deviation) during exposure was 21.5 (± 0.5), 21.4 (± 0.5), 21.4 (± 0.5) and 21.5 (± 0.5)°C, respectively. Measured relative humidity during exposure varied between 37.5 and 70.0%, i.e. within the range of 30-70% required by OECD guideline 412. For the control, low, mid and high concentration groups, mean relative humidity (± standard deviation) during exposure was 54.8 (± 4.5), 54.3 (± 5.5), 54.5 (± 5.3), and 54.8 (± 4.9)%, respectively.
- Air flow rate: The airflow was monitored by recording the settings of the mass view meter (group 1) and mass flow controllers (groups 2-4). During the range finding study, the total air flows were 16, 12, 16 and 16 L/min for the control, low, mid and high concentration exposure units, respectively. During the main study, total air flows were 32 L/min for all groups.
- Method of particle size determination: not applicable

TEST ATMOSPHERE
- Brief description of analytical method used: The actual concentration of anisole in the test atmosphere was determined by total carbon analysis. The test atmospheres were sampled from the exposure units at the animals’ breathing zone and passed to total carbon analyzers (TCA; Sick GmbH,Germany). The response of the analyzers was recorded on a pc using a CAN transmitter (G. Lufft Mess- und Regeltechnik GmbH, Felbach, Germany). The daily mean response was calculated by averaging values read every 0.5-1.5 minute. The nominal concentration was determined by dividing the total amount of test material used (by weight) by the total volume of air passed through the inhalation chamber. The generation efficiency was calculated from the actual and the nominal concentration.
- Samples taken from breathing zone: yes

VEHICLE: not applicable.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Actual concentration:
The mean actual concentrations (± standard deviation) as determined by total carbon analysis were 0.120 (± 0.001), 0.601 (± 0.005) and 3.01 (± 0.02) g/m3 for the low, mid and high concentration groups, respectively. These concentrations were very close to the respective target concentrations of 0.12, 0.6 and 3 g/m3 anisole.
- Nominal concentration and generation efficiency
The mean nominal concentrations (± standard deviation) were 0.124 (± 0.011), 0.629 (± 0.031) and 3.03 (± 0.06) g/m3 for the low, mid and high concentration groups, indicating generation efficiencies of 98%, 96% and 99%, respectively. The generation efficiencies were very close to 100%, as expected for generation of a vapour

Duration of treatment / exposure:

29 days

Frequency of treatment:

6 hours/day, 5 days/week

Doses / concentrations

Remarks:

Doses / Concentrations:
0 (control), 0.12, 0.6 and 3 g/m3
Basis:
analytical conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: The dose levels were chosen based on the results of a range-finding study
- Rationale for animal assignment (if not random): One day before the start of exposure, the animals were allocated to the various exposure groups by computer randomisation proportionally to body weight.
- Rationale for selecting satellite groups: no data
- Post-exposure recovery period in satellite groups: 18 days.
- Section schedule rationale (if not random): no data

Positive control:

No data

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. A group-wise observation was made halfway through each exposure day. On working days, all cages were checked again in the afternoon, especially for dead or moribund animals. In weekends and on public holidays only one check per day was carried out. During exposure however, observation was limited due to the animals’ stay in restraining tubes and attention was directed towards any breathing abnormalities and restlessness. All abnormalities, signs of ill health, and reactions to treatment were recorded.
- Cage side observations checked in table [No. 7.5.2/1] were included.

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal was recorded one day before the start of exposure and prior to exposure on the first day (day 0). Subsequently, animals of the range finding study were weighed on day 7; animals of the main study were weighed twice weekly for
the first two weeks (on days 4, 7, 11 and 14). Thereafter, the frequency was reduced to once weekly (i.e. on days 21 and 28), because there were no statistically significant effects on body weight in the first two weeks. Animals of the recovery groups were also weighed on days 35, 42 and 46. All animals were also weighed on their scheduled sacrifice date in order to calculate the correct organ to body weight ratios.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No. Food consumption was measured per cage by weighing the feeders.

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: NO

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: conducted at the end of the treatment period on all animals of the main study.
- Anaesthetic used for blood collection: No data
- Animals fasted: YES
- Parameters examined were:
- haemoglobin (Hb)
- packed cell volume (PCV)
- red blood cell count (RBC)
- reticulocytes
- total white blood cell count (WBC)
- differential white blood cell count(1)
- prothrombin time
- thrombocyte count
The following parameters were calculated:
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin (MCH)
- mean corpuscular haemoglobin concentration (MCHC)
(1) Lymphocytes, neutrophils, eosinophils, basophils and monocytes

Since changes were observed in prothrombin time in females of the high concentration group of the main study, blood samples were also collected from fasted male and female animals of the recovery groups to investigate prothrombin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: conducted at the end of the treatment period on all animals of the main study.
- Animals fasted: Yes
- Parameters examined were:
- alkaline phosphatase activity (ALP) - bilirubin total
- aspartate aminotransferase activity (ASAT) - cholesterol
- alanine aminotransferase activity (ALAT) - triglycerides
- gamma glutamyl transferase activity (GGT) - phospholipids
- total protein - calcium (Ca)
- albumin - sodium (Na)
- ratio albumin to globulin - potassium (K)
- urea - chloride (Cl)
- creatinine - inorganic phosphate
- fasting glucose

Since changes were observed in plasma levels of cholesterol, triglycerides, phospholipids, total protein, albumin and fasting glucose, these parameters were also investigated in animals of the recovery groups.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: conducted on all rats of all groups of the range finding study (on day -1 and prior to necropsy on day 7)
- Battery of functions tested: changes in skin and fur, piloerection, changes in the eyes, gait (including posture), and presence of clonic or tonic movements, stereotypies and bizarre behaviour.
Since treatment-related changes were not observed during the range finding study, neurobehavioural testing was not extended to the main study.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

- Estrus cycle evaluation:
Vaginal smears to evaluate the estrus cycle length and normality were made daily in the week prior to sacrifice, including the day of sacrifice, of all female animals of the main study. All smears were stained but – as no treatment-related abnormalities were observed. Only the smears of the control group and the high concentration group were evaluated. No vaginal smears were made of the female animals of the recovery groups.
- Sperm analysis: Epididymal sperm motility, count and morphology and Testicular sperm count.

Statistics:

‘Ancova & Dunnett’s Test’, Generalised Anova/Ancova Test’ and Fisher’s exact test.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical abnormalities were observed in response to exposure to Anisole.

Mortality:

no mortality observed

Description (incidence):

No treatment-related clinical abnormalities were observed in response to exposure to Anisole.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

observed at the high concentration level but not considered as treatment-related.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

observed at the high concentration level but not considered as treatment-related.

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):

observed at the high concentration level but reversible at the end of the 18-day recovery period

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

observed at the high concentration level but reversible at the end of the 18-day recovery period

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Since treatment-related changes were not observed during the range finding study, neurobehavioural testing was not extended to the main study.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

observed at the high concentration level but not considered as treatment-related.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortality was observed in this study. No treatment-related clinical abnormalities were observed in response to exposure to anisole. Sparsely haired skin was seen in one male and one female animal of the control group, one female animal of the low concentration group, one male animal of the mid concentration group and one male animal of the high concentration group. Encrustations of the skin were seen in one male animal of the control group. One male animal was found to miss part of a leg, and one female animal had a kink in the tail. These findings were not related to the exposure.

BODY WEIGHT AND WEIGHT GAIN
Significant changes in body weight did not occur during the exposure period, although a slight tendency towards decreased body weight gain was present in males of the high concentration group. During the recovery period, female animals of the high concentration group showed an increased body weight gain when compared to unexposed controls, which reached statistical significance on days 42 and 46.

FOOD CONSUMPTION
Slightly decreased consumption of food was observed in male animals of the high concentration group, which was most evident in the last week of the exposure. In female animals, food consumption was similar among the groups throughout the exposure period. During the recovery period, food consumption was slightly increased in female animals of the high concentration group.

HAEMATOLOGY
Prothrombin time was decreased in female animals of the high concentration group at the end of the exposure period. Such a decrease was no longer seen at the end of the recovery period. Other changes in red blood cell parameters or white blood cell counts were not found.

CLINICAL CHEMISTRY
Clinical chemistry results showed the following statistically significant differences between exposed animals and controls:

- Increased plasma glucose concentration in females of the high concentration group at the end of the exposure period. No changes in plasma glucose were observed in females at the end of the recovery period. In the absence of any changes in plasma glucose in males at the end of the exposure period, the slightly lower plasma glucose level in exposed males of the recovery group was considered likely to be a chance finding, probably the result of relatively high glucose levels in several of the current control animals.
- Increased plasma lipid concentrations in animals of the high concentration group at the end of the exposure period, which reached statistical significance in males with respect to cholesterol and phospholipids, and in females with respect to plasma triglycerides. No such changes in plasma lipid levels were observed in animals of the recovery group.
- Decreased albumin/globulin ratio in all female exposed groups at the end of the exposure period. However, in the absence of a clear dose-response relationship, any changes in total protein and albumin levels (used to calculate the albumin/globulin ratio) and any corroborative changes in males, this finding was considered to be of no toxicological relevance and likely to be the result of the current control values being at higher end of the historical control range. No changes in total protein, albumin or albumin/globulin ratio were observed in female animals of the recovery group.
- Decreased plasma level of total protein in exposed males of the recovery group.

NEUROBEHAVIOUR
Since treatment-related changes were not observed during the range finding study, neurobehavioural testing was not extended to the main study.

ORGAN WEIGHTS
At the end of the exposure period, absolute and relative weight of the liver weight and relative weight of the kidneys were significantly increased in male animals of the high concentration group when compared to controls. In female animals of the high concentration group, relative weight of the liver was also increased at the end of the exposure period. No changes in liver or kidney weight were observed at the end of the recovery period. The increased absolute and relative weight of the thymus in female animals of the low concentration group was not considered to be treatment-related,
since a dose-response relationship was not present. At the end of the recovery period, absolute and relative weight of the epididymis was decreased in male animals of the high concentration group when compared to unexposed controls. Relative brain weight was increased in males and decreased in females of the high concentration group at the end of the recovery period. However, these changes in relative brain weight were considered to be related to the body weight differences between exposed and control animals (lower and higher body weight in exposed males and females, compared to the respective controls), reflecting the independence of (absolute) brain weight from body weight. The lower relative heart and lung weights in female animals of the high concentration recovery group may also reflect the higher body weight of that group.

GROSS PATHOLOGY
Macroscopic examination at necropsy did not reveal any treatment-related pathological changes.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic examination of the sampled organs and tissues did not reveal any treatment-related histopathological changes. The histopathological changes observed were about equally distributed amongst the different treatment groups or occurred in one or a few animals only. They were common findings in rats of this strain and age or occurred as individual chance findings. Therefore, they were not considered to be related to the exposure to anisole.

OTHER FINDINGS (see Tables 7.5.2/ and 7.5.2/):
- Estrus cycle evaluation:
Vaginal smears obtained from females of the control and high concentration group during the last 7 days of the exposure period were evaluated. The number of acyclic females, the mean length of the longest cycle and the number of females with a prolonged estrus period were comparable in both groups.
- Sperm analysis:
* Epididymal sperm motility:
No statistically significant differences between the control and exposed groups were observed with respect to sperm motility parameters at the end of the exposure period.
* Epididymal sperm count:
No statistically significant changes in epididymal sperm count were observed in response to exposure to anisole.
* Testicular sperm count:
No statistically significant changes in the number of spermatozoa per gram testicular parenchyma or in daily sperm production were observed in response to exposure to anisole.
* Epididymal sperm morphology:
Smears of one control and one animal of the high concentration group could not be judged, because the number of cells was too low. The slides of another control and an animal of the high concentration group contained relatively high numbers of cells that were damaged during the procedure, which made it difficult to distinguish malformed from damaged cells. Nevertheless, a slight – but statistically significant – increase in the number of large hooked cells was observed in animals of the high concentration group relative to unexposed controls. However, since only a very small absolute number of this abnormal cell type was observed, and all other morphology parameters were unaffected upon exposure to anisole, the small
increase in the number of large hooked cells was considered to be a chance finding.

Effect levels

1

Dose descriptor:

NOAEC

Effect level:

3 000 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOEC

Effect level:

600 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No local or systemic effects were observed at 0.6 or 0.12 g/m3.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 7.5.2/2: Mean haematology results: red blood cell and coagulation parameters (at the end of the treatment period + recovery results)/Males

Sex: Male		RBC (10E12/L) GEN AN (AUTO)	Hb (mmol/L) GEN AN (AUTO)	PCV (L/L) GEN AN (AUTO)	MCV (fL) GEN AN (AUTO)	MCH (fmol) GEN AN (AUTO)	MCHC (mmol/L) GEN AN (AUTO)	Reticulocytes (%) GEN AN (AUTO)	Thrombocytes (10E9/L) GEN AN (AUTO)	ProthromTime(s) GEN AN (AUTO)	ProthromTime(s) GEN AN (AUTO)
Control	Mean SD N	8.296* 0.592 5	9.48* 0.40 5	0.4420* 0.0281 5	53.32* 1.84 5	1.146* 0.064 5	21.48 0.73 5	2.278* 0.470 5	1031.4* 72.0 5	38.82* 2.92 5	45.84* 7.44 5
0.12 g/m3	Mean SD N	8.266 0.575 5	9.32 0.44 5	0.4314 0.0240 5	52.24 1.29 5	1.129 0.037 5	21.62 0.54 5	2.028 0.300 5	1151.2 141.0 5	37.26 1.05 5	-
0.6 g/m3	Mean SD N	8.174 0.373 5	9.66 0.23 5	0.4360 0.0099 5	53.39 1.67 5	1.184 0.066 5	22.16 0.54 5	2.040 0.281 5	1030.2 59.4 5	39.22 2.37 5	-
3 g/m3	Mean SD N	8.206 0.391 5	9.62 0.36 5	0.4410 0.0150 5	53.82 2.77 5	1.175 0.078 5	21.82 0.40 5	2.068 0.242 5	1098.2 85.6 5	36.00 1.66 5	44.20 6.37 5

Sex : Male		WBC (10^E9/L) GEN AN(AUTO)	Lympho Absolute (10^E9/L) GEN AN(AUTO)	Neutro Absolute (10^E9/L) GEN AN(AUTO)	Eosino Absolute (10^E9/L) GEN AN(AUTO)	Baso Absolute (10^E9/L) GEN AN(AUTO)	Mono Absolute (10^E9/L) GEN AN(AUTO)
Control	Mean SD N	4.56* 0.92 5	3.58* 0.74 5	0.83* 0.31 5	0.058** 0.027 5	0.003* 0.002 5	0.088* 0.031 5
0.12 g/m3	Mean SD N	3.62 0.8 5	2.73 0.68 5	0.76 0.20 5	0.042 0.016 5	0.002 0.002 5	0.086 0.022 5
0.6 g/m3	Mean SD N	4.54 1.18 5	3.62 1.00 5	0.78 0.18 5	0.050 0.011 5	0.006 0.004 5	0.084 0.053 5
3 g/m3	Mean SD N	5.26 0.4 5	4.14 0.57 5	0.94 0.24 5	0.048 0.006 5	0.006 0.003 5	0.124 0.048 5
		Lymphocytes (%) GEN AN(AUTO)	Neutrophils (%) GEN AN(AUTO)	Eosinophils (%) GEN AN(AUTO)	Basophils (%) GEN AN(AUTO)	Monocytes (%) GEN AN(AUTO)
Control	Mean SD N	78.52* 5.40 5	18.20* 4.58 5	1.24** 0.45 5	0.06* 0.05 5	1.96** 0.79 5
0.12 g/m3	Mean SD N	75.34 4.86 5	21.04 4.58 5	1.14 0.30 5	0.06 0.05 5	2.40 0.52 5
0.6 g/m3	Mean SD N	79.40 3.97 5	17.54 3.52 5	1.12 0.15 5	0.12 0.08 5	1.80 0.79 5
3 g/m3	Mean SD N	78.54 6.38 5	18.06 5.49 5	0.92 0.08 5	0.12 0.04 5	2.40 1.08 5

*: Automatic transformation

**: Automatic transformation

Table 7.5.2/3: Mean haematology results: red blood cell and coagulation parameters (at the end of the treatment period + recovery results)/Females

Sex: Female		RBC (10E12/L) GEN AN (AUTO)	Hb (mmol/L) GEN AN (AUTO)	PCV (L/L) GEN AN (AUTO)	MCV (fL) GEN AN (AUTO)	MCH (fmol) GEN AN (AUTO)	MCHC (mmol/L) GEN AN (AUTO)	Reticulocytes (%) GEN AN (AUTO)	Thrombocytes (10E9/L) GEN AN (AUTO)	ProthromTime(s) GEN AN (AUTO)	ProthromTime(s) GEN AN (AUTO)
Control	Mean SD N	7.894* 0.280 5	8.86* 0.23 5	0.4106* 0.0100 5	52.04* 1.03 5	1.123* 0.048 5	21.58* 0.51 5	2.018* 0.402 5	1081.2** 45.3 5	34.92** 2.42 5	34.70* 2.91 5
0.12 g/m3	Mean SD N	7.616 0.204 5	9.08 0.19 5	0.4100 0.0105 5	53.84 0.99 5	1.193 0.042 5	22.15 0.46 5	1.866 0.156 5	1159.8 111.8 5	33.50 2.91 5	-
0.6 g/m3	Mean SD N	8.012 0.215 5	9.24 0.34 5	0.4230 0.0082 5	52.81 0.80 5	1.153 0.038 5	21.84 0.56 5	1.884 0.310 5	995.6 92.1 5	33.66 1.18 5	-
3 g/m3	Mean SD N	7.780 0.487 5	8.92 0.18 5	0.4098 0.0157 5	52.75 1.78 5	1.150 0.074 5	21.79 0.72 5	2.186 0.292 5	1051.4 71.6 5	30.92 d*** 1.28 5	33.80 3.32 5

Sex : Female		WBC (10^E9/L) GEN AN(AUTO)	Lympho Absolute (10^E9/L) GEN AN(AUTO)	Neutro Absolute (10^E9/L) GEN AN(AUTO)	Eosino Absolute (10^E9/L) GEN AN(AUTO)	Baso Absolute (10^E9/L) GEN AN(AUTO)	Mono Absolute (10^E9/L) GEN AN(AUTO)
Control	Mean SD N	2.94* 0.84 5	2.57** 0.83 5	0.30 R3 0.03 5	0.026* 0.009 5	0.001* 0.001 5	0.051* 0.019 5
0.12 g/m3	Mean SD N	3.32 0.83 5	2.89 0.74 5	0.35 0.09 5	0.027 0.007 5	0.003 0.002 5	0.055 0.021 5
0.6 g/m3	Mean SD N	2.66 0.62 5	2.23 0.65 5	0.35 0.07 5	0.024 0.004 5	0.001 0.002 5	0.050 0.014 5
3 g/m3	Mean SD N	2.66 0.62 5	2.23 0.65 5	0.35 0.07 5	0.024 0.004 5	0.001 0.002 5	0.050 0.014 5
		Lymphocytes (%) GEN AN(AUTO)	Neutrophils (%) GEN AN(AUTO)	Eosinophils (%) GEN AN(AUTO)	Basophils (%) GEN AN(AUTO)	Monocytes (%) GEN AN(AUTO)
Control	Mean SD N	86.54* 3.65 5	10.78* 3.54 5	0.92* 0.27 5	0.04** 0.05 5	1.74* 0.54 5
0.12 g/m3	Mean SD N	86.88 1.94 5	10.56 1.62 5	0.84 0.17 5	0.08 0.04 5	1.64 0.50 5
0.6 g/m3	Mean SD N	85.48 2.62 5	11.54 2.23 5	0.74 0.23 5	0.06 0.05 5	2.20 0.40 5
3 g/m3	Mean SD N	83.20 5.03 5	13.86 4.32 5	0.96 0.32 5	0.04 0.09 5	1.92 0.58 5

*: Automatic transformation: Identity (None)

d***: Test Dunnett 2 sided p <0.05

**: Automatic transformation: Identity (None), Group Factor Test: Anova p <0.05

R3: Automatic transformation: Rank

 

Table 7.5.2/4: Mean clinical chemistry results (at the end of the treatment period)/Males

Sex : Male		ALP (U/L) GEN AN(AUTO)	ASAT (U/L) GEN AN(AUTO)	ALAT (U/L) GEN AN(AUTO)	CGT (U/L) GEN AN(AUTO)	Bilirub Total (umol/L) GEN AN(AUTO)	Total Protein (g/l) GEN AN(AUTO)	Albumin GEN AN(AUTO)
Control	Mean SD N	123.8* 54.9 5	91.2** 23.8 5	49.6*** 7.8 5	0.34*** 0.41 5	1.98*** 0.54 5	59.2*** 1.1 5	30.8*** 0.4 5
0.12 g/m3	Mean SD N	121.2 20.4 5	90.8 19.4 5	55.6 15.2 5	0.28 0.26 5	1.96 0.29 5	60.2 2.2 5	31.8 1.3 5
0.6 g/m3	Mean SD N	162.4 45.3 5	101.2 18.0 5	51.8 8.4 5	0.46 0.46 5	2.06 0.34 5	61.2 1.6 5	31.8 0.8 5
3 g/m3	Mean SD N	109.2 27.2 5	97.6 53.6 5	56.0 11.2 5	0.48 0.33 5	1.80 0.30 5	62.4 2.8 5	31.6 0.9 5
		Albumin/Globulin GEN AN(AUTO)	Glucose Plasma (mmol/l)   GEN AN(AUTO)	Cholesterol (mmol/l)	Phospholipids (mmol/l) GEN AN(AUTO)	Triglycerides (mmol/l)   GEN AN(AUTO)	Creatinine GEN AN(AUTO)	Urea (mmol/L)   GEN AN(AUTO)
Control	Mean SD N	1.086*** 0.050 5	7.294*** 1.061 5	1.578 0.129 5	1.35 0.08 5	0.320 0.096 5	28.0* 2.6 5	7.92 0.55 5
0.12 g/m3	Mean SD N	1.122 0.071 5	8.044 0.703 5	1.778 0.11 5	1.49 0.10 5	0.458 0.196 5	26.2 4.0 5	7.28 0.91 5
0.6 g/m3	Mean SD N	1.083 0.041 5	7.762 1.324 5	1.58 0.252 5	1.34 0.14 5	0.382 0.108 5	26.2 2.6 5	7.46 1.49 5
3 g/m3	Mean SD N	1.028 0.036 5	7.454 1.313 5	2.084dd2 0.301 5	1.61d4 0.19 5	0.542 0.070 5	27.0 3.2 5	8.08 0.67 5

 *: Automatic transformation: Log

**: Automatic transformation: Rank

***: Automatic transformation: Identity (none)

Table 7.5.2/5: Mean clinical chemistry results (at the end of the recovery period)/Males

Sex : Male		Total protein (g/L)	Albumin (g/L)	Albumin/Globulin	Glucose plasma (mmol/L)	Cholesterol (mmol/L)	Phospholipids (mmol/L)	Triglycerides (mmol/L)
Control	Mean SD N	60.6I, a1 2.1 5	31.4 I3 1.1 5	1.079 I3 0.076 5	7.942I, a1 1.664 5	1.694 I3 0.160 5	1.500 I3 0.144 5	0.664 l4 0.174 5
3 g/m3	Mean SD N	57.6 d2 1.1 5	30.4 1.1 5	1.119 0.065 5	5.724 d2 1.058 5	1.490 0.220 5	1.336 0.204 5	0.558 0.406 5

1 I,a-Automatic Transformation: identity (none), Group Factot test: Anova p <0.05

3 I-Automatic transformation: Identity (none)

2 d-Test: Dunnett 2 Sided P <0.05

4 L-Automatic transformation: Log

 

Applicant's summary and conclusion

Conclusions:

Exposure to anisole resulted in treatment-related changes in the high concentration group only. Most changes were reversible and were not accompanied by any histopathological changes. Therefore, the No-Observed-Adverse-Effect-Level (NOAEL) in rats exposed for 6 hours/day, 5 days/week for a period of 29 days (20 exposure days) was 3 g/m3 under the test conditions of this study.

Executive summary:

The inhalation toxicity of anisole was studied in a sub-acute (29-day) inhalation toxicity study in Wistar rats according to the OECD guideline N°412 and in compliance with GLP. Four groups of 5 male and 5 female rats were exposed nose-only to target concentrations of 0 (control), 0.12, 0.6 or 3g/m3 for 6 hours/day, 5 days/week over a 29-day period, with a total of 20 exposure days. An additional control and high concentration group of 5 male and 5 female rats each were exposed similarly to 0 or 3g/m3 respectively, and kept for a recovery period of 18 days after the last exposure. Clinical observations, body weight, food consumption, haematology and clinical chemistry were collected. Estrus cyclicity was evaluated during the last week of the exposure period. At necropsy, the animals were examined for gross macroscopic abnormalities, organs were weighed, sperm parameters – numbers, motility and morphology – were analyzed, and a selection of organs and tissues (including the complete respiratory tract with nasal passages) was examined microscopically.

The concentration levels for the 29-day main study were based on the results of a 7-day range finding study.

No treatment-related clinical abnormalities or mortality were observed in response to the exposure to anisole. Relevant changes in toxicological endpoints were found at the high concentration level only. Relative liver weight was increased by approximately 15% in both male and female animals. Relative kidney weight was also slightly increased in males of the high concentration group. These organ weight changes were accompanied by increased plasma glucose concentrations in females and increased plasma lipid concentrations (cholesterol, phospholipids and triglycerides) in males and females of the high concentration group. However, histopathological changes in the liver were not observed and all of the above mentioned changes appeared reversible at the end of the 18-day recovery period. Other changes in the high concentration group, including a slight tendency towards growth retardation and food consumption in males and a slightly decreased protrombin time in females, also proved to be reversible within the recovery period. At the end of the exposure period, absolute liver weight was increased in males of the high concentration group, and relative (to body weight) liver weight was increased in males and females of the high concentration group. In addition, relative kidney weight was increased in males of the high concentration group. No changes in liver or kidney weight were observed at the end of the recovery period. Although not observed at the end of the exposure period, absolute and relative weight of the epididymis was increased in exposed males at the end of the recovery period. Other organ weight changes in exposed animals of the recovery group included increased relative brain weight in males, and decreased relative brain, lung and heart weight in females. These changes were considered likely to be related to body weight differences between exposed and control animals.

Analysis of reproduction parameters did not reveal any treatment-related abnormalities with respect to estrus cyclicity, sperm numbers, motility or sperm morphology.

Macroscopic examination at necropsy and histopathological examination of organs and tissues – including the complete respiratory tract – did not reveal any treatment-related changes.

 

In conclusion, exposure to anisole resulted in treatment-related changes in the high concentration group only. Most changes were reversible and were not accompanied by any histopathological changes. Therefore, the No-Observed-Adverse-Effect-Level (NOAEL) in rats exposed for 6 hours/day, 5 days/week for a period of 29 days (20exposure days) was 3000 mg/m3 under the test conditions of this study.

This sub- acute study is classified as acceptable. It satisfies the guideline requirement for an inhalation sub-acute toxicity study in rat.