Registration Dossier

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to OECD guidelines to to GLP, and therefore meets the requirements for Klimisch code 1.

Guideline:

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

Deviations:

yes

Remarks:

A fuctional observation battery for neurotoxicity was not performed since this test was not part of the OECD 407 guideline at the time the study was performed

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 41 days
- Weight at study initiation: males, 179-215g; female 141-170g
- Housing: hanging stainless steel wire-bottom cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-23°C
- Humidity (%): 48-66%
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

Route of administration:

oral: gavage

Vehicle:

peanut oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Mixed weekly weight/volume in peanut oil

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analysis of dosing solutions was conducted to confirm that they were homogeneous and met the desired concentrations.

Duration of treatment / exposure:

29 day treatment duration with a 14 day recovery period.

Frequency of treatment:

7 days/week.

Remarks:

Doses / Concentrations:
gavage doses of 0, 100, 500 or 1000 mg/kg/bw/day
Basis:
actual ingested

No. of animals per sex per dose:

6

Control animals:

yes

Details on study design:

- Dose selection rationale: Data from a pilot two week repeated dose oral study

Positive control:

No.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Twice weekly

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
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

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
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Weekly
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At termination
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: All

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At termination
- Animals fasted: Yes
- How many animals: All

URINALYSIS: Yes
- Time schedule for collection of urine: Overnight before termination
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

Tests applied included; parametric ANOVA with Dunnetts post-hoc test, non parametric Kruskal-Wallis and Mann-Whitney U test, Bartletts test for equal variances, Students t test and Dixons test for rejection of outlying values

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
One animal was sacrificed on Day 0 and one animal was found dead on Day 9, a result of probable misdosing.

Stained fur was observed in high dose animals, scabbed skin occurred in one control male and high dose female displayed sneezing and abnormal respiratory sounds.

BODY WEIGHT AND WEIGHT GAIN
No statistically significant differences were observed in mean bodyweights or body weight gains.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
A statistically significant increase in food consumption was observed in low dose males compared with controls.

FOOD EFFICIENCY
No statistically significant differences were observed in food efficiency.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
Not recorded

OPHTHALMOSCOPIC EXAMINATION
Not recorded

HAEMATOLOGY
Male mean cell haemoglobin concentrations were significantly decreased compared with the controls at all dose levels. However, these were not considered to be biologically significant, as there was no dose response trend. A statistically significant increase in partial thromboplastin time was observed in mid and top dose males compared with controls. Prothrombin time was significantly increased in the mid and high dose females during the treatment period, and was significantly reduced in males in the recovery group. This were within normal limits and therefore not considered to be biologically significant. A statistically significant increase in the reticulocyte count was observed in treated males in the recovery group, however, was not considered to be biologically significant.

CLINICAL CHEMISTRY
A statistically significant decrease in serum cholesterol was observed in high dose males and females and persisted in females into the recovery period. This was considered to be treatment related.

Statistically significant increases were observed in alanine aminotransferase, lactic dehydrogenase, aspartate aminotransferase, sodium, phosphorus and triglycerides, as well as decreases in albumin and chloride.There was no dose related trend with these changes, therefore they are not considered to be treatment related.

URINALYSIS
A statistically significant increase in specific gravity was observed in low dose males. Urine volume was significantly reduced in treated males in the recovery group. This was not considered to be biologically significant.

NEUROBEHAVIOUR
Not recorded

ORGAN WEIGHTS
No statistically significant differences were observed.

GROSS PATHOLOGY
No substance related macroscopic changes were observed.

HISTOPATHOLOGY: NON-NEOPLASTIC
No substance related microscopic changes were observed.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
Not recorded

HISTORICAL CONTROL DATA (if applicable)
Not recorded

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: Mean serum cholesterol levels were significantly reduced in the 1000 mg/kg males and females at termination of dosing. This was still significantly reduced in 1000 mg/kg females at the end of the 14 day recovery period.

Critical effects observed:

not specified

Table 1: Average body weights and body weight gains during xx days of treatment

Dose rate (ppm)	Body Weights (g)					Total Weight Gain
Dose rate (ppm)	Week 0	Week 1	Week 2	Week 3	Week 4	g	% of control
Male
0	195	243	286	323	352	157	181
100	196	245	298	340	374	175	191
500	200	245	289	329	362	162	181
1000	193	240	283	315	346	154	179
Female
0	155	175	194	213	223	67	144
100	156	174	194	215	228	72	146
500	154	175	190	212	221	67	144
1000	155	174	195	212	224	69	145

Conclusions:

A NOAEL of 500 mg/kg bw/day was identified in this study.

Executive summary:

In a subchronic toxicity study calcium sulphonate was administered to 12 rats/sex in the control and top dose and 6 rats/sex in the low and mid dose via gavage at dose levels of 0, 100, 500 or 1000 mg/kg bw/day.

A decrease in serum cholesterol levels occurred in the top dose group. The LOAEL is 1000 mg/kg bw/day, based on a decrease in serum cholesterol at the top dose. The NOAEL is 500 mg/kg bw/day.

This subchronic toxicity study in the rat is acceptable and satisfies the guideline requirement for a subchronic oral study (OPPTS 870.3100; OECD 408) in rats.

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subacute

Species:

rat

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to OECD guidelines and to GLP, and therefore meets the criteria for Klimisch code 1.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River
- Age at study initiation: males, 6 weeks; females, 7 weeks
- Weight at study initiation: males, 205-232g; females 156-186g
- Housing: suspended wire mesh cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-26°C
- Humidity (%): 20-76%
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: MMAD 3.3-3.7 µm
GSD 2.0-2.1 µm

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:Glass and stainless steel exposure chambers
- System of generating particulates/aerosols:
- Temperature, humidity, pressure in air chamber: 21-26°C, 20-76%
- Air flow rate: 210-215 pm
- Air change rate: 4.8/minute
- Method of particle size determination: Delron DCI-6 cascade impactor

TEST ATMOSPHERE
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Concentration measured by gravimetric analysis.

Duration of treatment / exposure:

6 hours per day for 28 days.

Frequency of treatment:

5 days per week.

Remarks:

Doses / Concentrations:
49.5, 156, 260 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent no treatment

Positive control:

None.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

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: Prior to terminal sacrifice
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: All animals
- Parameters examined: Haemoglobin concentration, haematocrit, erythrocyte count, platelet count, clotting time, total and differential leukocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to terminal sacrifice
- Animals fasted: No data
- How many animals: All animals

URINALYSIS: Yes
- Time schedule for collection of urine: Prior to terminal sacrifice
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

gross pathology on all animals
GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)

Statistics:

ANOVA with Dunnets test
Regression analysis
Krusal-Walis and Dunns summed rank test
Jonsheere's test for monotonic trend

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortality occurred during the test.

BODY WEIGHT AND WEIGHT GAIN
Bodyweight gain was decreased in treated animals, although this was not statistically significant.

FOOD CONSUMPTION
Not measured

FOOD EFFICIENCY
Not measured

WATER CONSUMPTION
Not measured

OPHTHALMOSCOPIC EXAMINATION
Not measured

HAEMATOLOGY
Statistically significant differences were observed in females. Specifically; increased haematocrit in the low dose group.
CLINICAL CHEMISTRY
Statistically significant differences were observed in females. Specifically; increased creatine phospholinase in the mid and top dose groups and sodium in the top dose group.

NEUROBEHAVIOUR
Not measured

ORGAN WEIGHTS
Statistically significant increased lung weights and lung to body weight ratios were observed in a dose dependant manner in the mid and top dose groups.

GROSS PATHOLOGY
Gross lesions were sporadic and were not considered to be caused by the test article.

HISTOPATHOLOGY: NON-NEOPLASTIC
A higher incidence of accumulations of intraalveolar macrophages and hyperplasia/hypertrophy of bronchiole epithelium was seen in the lungs of the treated animals. These were dose related at the mid and top dose levels.

Dose descriptor:

NOAEL

Effect level:

49.5 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: Statistically significant dose related increase in lung weight and relative lung weights with corresponding accumulations of intraalveolar macrophages and hyperplasia/hypertrophy of the bronchiole epithelium.

Critical effects observed:

not specified

Table 1: Average body weights and body weight gains during 28 days of treatment

Analytical concentration (mg/L)	Body Weights (g)					Total Weight Gain
Analytical concentration (mg/L)	Week 0	Week 1	Week 2	Week 3	Week 4	g	% of control
Male
0	217	249	295	336	369	152
LCT	216	248	294	331	359	143	94
MCT	215	247	302	346	384	169	111
HCT	215	243	285	319	347	132	87
Female
0	171	185	206	223	239	68
LCT	169	185	205	220	232	63	93
MCT	168	181	203	219	229	61	90
HCT	172	186	205	223	238	66	97

Table 2 Selected haematology, clinical chemistry and pathology findings

Doses (unit)	0	50	150	250	0	50	150	250
Doses (unit)	male				female
Number of animals/group	5	5	5	5	5	5	5	5
Haematology(day x)
- RBC (TERA/L)	6.62	6.89	6.66	6.52	6.60	6.90	6.59	6.70
- MCV (FL)	-	-	-	-	-	-	-	-
- HCT (L/L)	46	47	46	43	46	48	47	44
- HGB (MMOL/L)	16.1	16.7	16.1	15.5	15.5	16.6	16.1	15.5
- WBC (GIGA/L)	13.9	13.7	12.9	10.5	9.0	12.8	11.5	13.8
Blood chemistry(day x)
- sodium (MMOL/L)	146	146	147	146	145	143	144	141
- potassium (MMOL/L)	4.4	4.4	4.5	4.3	4.3	4.3	4.3	4.3
- chloride (MMOL/L)	101	102	102	102	102	102	102	101
- gobulin (G/L)	2.2	2.3	2.4	2.0	2.1	2.1	2.1	2.0
- cholesterol (MMOL/L)	56	53	63	55	59	75	60	69
- triglyceride (MMOL/L)	45	39	54	35	30	24	25	25
Pathology
Accumulation of intraalveolar macrophages	3	5	5	5	5	5	5	5
Bronchiolar epithelium: hyperplasia/hypertrophy	3	4	5	5	4	5	5	5

Table 3: Absolute and relative organ weights

		Males				Females
DAILY DOSE (units e.g. mg/kg bw/day)		0	50	150	250	0	50	150	250
NUMBER OF ANIMALS		5	5	5	5	5	5	5	5
BODY WEIGHT (g)^a		335	323	346	313	213	204	204	208
BRAIN
Absolute Weight^a	g	1.976	1.939	2.032	1.943	1.900	1.876	1.856	1.830
Per Body Weight^a	%	5.93	6.03	5.90	6.21	8.96	9.20	9.12	8.84
ADRENALS
Absolute Weight^a	g	0.051	0.052	0.054	0.054	0.068	0.069	0.070	0.060
Per Body Weight^a	%	1.52	1.63	1.56	1.71	3.21	3.37	3.44	2.90
Per Brain Weight^a	%
HEART
Absolute Weight^a	g	1.220	1.173	1.179	1.122	0.803	0.801	0.753	0.793
Per Body Weight^a	%	3.64	3.65	3.41	3.59	3.79	3.92	3.70	3.82
Per Brain Weight^a	%
KIDNEYS
Absolute Weight^a	g	2.592	2.779	3.054	2.704	1.745	1.696	1.694	1.883
Per Body Weight^a	%	7.79	8.61	8.81	8.61	8.16	8.32	8.29	9.05
Per Brain Weight^a	%
LIVER
Absolute Weight^a	g	10.775	10.306	11.470	9.999	7.020	6.464	6.789	7.357
Per Body Weight^a	%	3.22	3.20	3.31	3.18	3.30	3.17	3.33	3.53
Per Brain Weight^a	%
SPLEEN
Absolute Weight^a	g	0.670	0.569	0.652	0.594	0.426	0.441	0.471	0.484
Per Body Weight^a	%	2.00	1.77	1.88	1.89	2.01	2.17	2.30	2.34
Per Brain Weight^a	%
TESTES						n.a.^b	n.a.^b	n.a.^b	n.a.^b
Absolute Weight^a	g	3.187	3.072	2.796	3.135	n.a.^b	n.a.^b	n.a.^b	n.a.^b
Per Body Weight^a	%	9.54	9.54	8.16	10.02	n.a.^b	n.a.^b	n.a.^b	n.a.^b
Per Brain Weight^a	%					n.a.^b	n.a.^b	n.a.^b	n.a.^b
LUNGS
Absolute Weight^a	g	1.306	1.302	1.515	1.537	1.051	1.127	1.138	1.338
Per Body Weight^a	%	3.91	4.05	4.39	4.91	4.93	5.52	5.59	6.46
Per Brain Weight^a	%
OVARIES		n.a.^b	n.a.^b	n.a.^b	n.a.^b	0.0921	0.0768	0.0943	0.0742
Absolute Weight^a	g	n.a.^b	n.a.^b	n.a.^b	n.a.^b	4.34	3.77	4.62	3.56
Per Body Weight^a	%	n.a.^b	n.a.^b	n.a.^b	n.a.^b
Per Brain Weight^a	%	n.a.^b	n.a.^b	n.a.^b	n.a.^b

Conclusions:

A NOAEL of 49.5 mg/m³ was identified for males and females, based on increased lung weight and microscopic changes of the lung.

Executive summary:

In a subacute inhalation toxicity study, a petroleum derived calcium salt was administered to 5 Sprague-Dawley rats/sex/concentration by whole body exposure at concentrations of 0, 49.5, 156 or 260 mg/m³ for 6 hours per day, 5 days/week for a total of 28 days.

Statistically significant, dose related increases in lung weights occurred in the mid and top dose groups, with corresponding increases in intraalveolar macrophages and hyperplasia/hypertrophy of bronchiole epithelium. The LOAEL is 156 mg/m³, based on effects in the lung. The NOAEL is 49.5 mg/m³.

This subchronic toxicity study in the rat is acceptable and satisfies the guideline requirement (OECD 412) for a subchronic inhalation study in the rat.

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

881.58 mg/m³

Study duration:

subacute

Species:

rat

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to OECD guidelines and to GLP, and therefore meets the criteria for Klimisch code 1.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River
- Age at study initiation: males, 6 weeks; females, 7 weeks
- Weight at study initiation: males, 205-232g; females 156-186g
- Housing: suspended wire mesh cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-26°C
- Humidity (%): 20-76%
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: MMAD 3.3-3.7 µm
GSD 2.0-2.1 µm

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:Glass and stainless steel exposure chambers
- System of generating particulates/aerosols:
- Temperature, humidity, pressure in air chamber: 21-26°C, 20-76%
- Air flow rate: 210-215 pm
- Air change rate: 4.8/minute
- Method of particle size determination: Delron DCI-6 cascade impactor

TEST ATMOSPHERE
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Concentration measured by gravimetric analysis.

Duration of treatment / exposure:

6 hours per day for 28 days.

Frequency of treatment:

5 days per week.

Remarks:

Doses / Concentrations:
49.5, 156, 260 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent no treatment

Positive control:

None.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

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: Prior to terminal sacrifice
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: All animals
- Parameters examined: Haemoglobin concentration, haematocrit, erythrocyte count, platelet count, clotting time, total and differential leukocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to terminal sacrifice
- Animals fasted: No data
- How many animals: All animals

URINALYSIS: Yes
- Time schedule for collection of urine: Prior to terminal sacrifice
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

gross pathology on all animals
GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)

Statistics:

ANOVA with Dunnets test
Regression analysis
Krusal-Walis and Dunns summed rank test
Jonsheere's test for monotonic trend

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortality occurred during the test.

BODY WEIGHT AND WEIGHT GAIN
Bodyweight gain was decreased in treated animals, although this was not statistically significant.

FOOD CONSUMPTION
Not measured

FOOD EFFICIENCY
Not measured

WATER CONSUMPTION
Not measured

OPHTHALMOSCOPIC EXAMINATION
Not measured

HAEMATOLOGY
Statistically significant differences were observed in females. Specifically; increased haematocrit in the low dose group.
CLINICAL CHEMISTRY
Statistically significant differences were observed in females. Specifically; increased creatine phospholinase in the mid and top dose groups and sodium in the top dose group.

NEUROBEHAVIOUR
Not measured

ORGAN WEIGHTS
Statistically significant increased lung weights and lung to body weight ratios were observed in a dose dependant manner in the mid and top dose groups.

GROSS PATHOLOGY
Gross lesions were sporadic and were not considered to be caused by the test article.

HISTOPATHOLOGY: NON-NEOPLASTIC
A higher incidence of accumulations of intraalveolar macrophages and hyperplasia/hypertrophy of bronchiole epithelium was seen in the lungs of the treated animals. These were dose related at the mid and top dose levels.

Dose descriptor:

NOAEL

Effect level:

49.5 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: Statistically significant dose related increase in lung weight and relative lung weights with corresponding accumulations of intraalveolar macrophages and hyperplasia/hypertrophy of the bronchiole epithelium.

Critical effects observed:

not specified

Table 1: Average body weights and body weight gains during 28 days of treatment

Analytical concentration (mg/L)	Body Weights (g)					Total Weight Gain
Analytical concentration (mg/L)	Week 0	Week 1	Week 2	Week 3	Week 4	g	% of control
Male
0	217	249	295	336	369	152
LCT	216	248	294	331	359	143	94
MCT	215	247	302	346	384	169	111
HCT	215	243	285	319	347	132	87
Female
0	171	185	206	223	239	68
LCT	169	185	205	220	232	63	93
MCT	168	181	203	219	229	61	90
HCT	172	186	205	223	238	66	97

Table 2 Selected haematology, clinical chemistry and pathology findings

Doses (unit)	0	50	150	250	0	50	150	250
Doses (unit)	male				female
Number of animals/group	5	5	5	5	5	5	5	5
Haematology(day x)
- RBC (TERA/L)	6.62	6.89	6.66	6.52	6.60	6.90	6.59	6.70
- MCV (FL)	-	-	-	-	-	-	-	-
- HCT (L/L)	46	47	46	43	46	48	47	44
- HGB (MMOL/L)	16.1	16.7	16.1	15.5	15.5	16.6	16.1	15.5
- WBC (GIGA/L)	13.9	13.7	12.9	10.5	9.0	12.8	11.5	13.8
Blood chemistry(day x)
- sodium (MMOL/L)	146	146	147	146	145	143	144	141
- potassium (MMOL/L)	4.4	4.4	4.5	4.3	4.3	4.3	4.3	4.3
- chloride (MMOL/L)	101	102	102	102	102	102	102	101
- gobulin (G/L)	2.2	2.3	2.4	2.0	2.1	2.1	2.1	2.0
- cholesterol (MMOL/L)	56	53	63	55	59	75	60	69
- triglyceride (MMOL/L)	45	39	54	35	30	24	25	25
Pathology
Accumulation of intraalveolar macrophages	3	5	5	5	5	5	5	5
Bronchiolar epithelium: hyperplasia/hypertrophy	3	4	5	5	4	5	5	5

Table 3: Absolute and relative organ weights

		Males				Females
DAILY DOSE (units e.g. mg/kg bw/day)		0	50	150	250	0	50	150	250
NUMBER OF ANIMALS		5	5	5	5	5	5	5	5
BODY WEIGHT (g)^a		335	323	346	313	213	204	204	208
BRAIN
Absolute Weight^a	g	1.976	1.939	2.032	1.943	1.900	1.876	1.856	1.830
Per Body Weight^a	%	5.93	6.03	5.90	6.21	8.96	9.20	9.12	8.84
ADRENALS
Absolute Weight^a	g	0.051	0.052	0.054	0.054	0.068	0.069	0.070	0.060
Per Body Weight^a	%	1.52	1.63	1.56	1.71	3.21	3.37	3.44	2.90
Per Brain Weight^a	%
HEART
Absolute Weight^a	g	1.220	1.173	1.179	1.122	0.803	0.801	0.753	0.793
Per Body Weight^a	%	3.64	3.65	3.41	3.59	3.79	3.92	3.70	3.82
Per Brain Weight^a	%
KIDNEYS
Absolute Weight^a	g	2.592	2.779	3.054	2.704	1.745	1.696	1.694	1.883
Per Body Weight^a	%	7.79	8.61	8.81	8.61	8.16	8.32	8.29	9.05
Per Brain Weight^a	%
LIVER
Absolute Weight^a	g	10.775	10.306	11.470	9.999	7.020	6.464	6.789	7.357
Per Body Weight^a	%	3.22	3.20	3.31	3.18	3.30	3.17	3.33	3.53
Per Brain Weight^a	%
SPLEEN
Absolute Weight^a	g	0.670	0.569	0.652	0.594	0.426	0.441	0.471	0.484
Per Body Weight^a	%	2.00	1.77	1.88	1.89	2.01	2.17	2.30	2.34
Per Brain Weight^a	%
TESTES						n.a.^b	n.a.^b	n.a.^b	n.a.^b
Absolute Weight^a	g	3.187	3.072	2.796	3.135	n.a.^b	n.a.^b	n.a.^b	n.a.^b
Per Body Weight^a	%	9.54	9.54	8.16	10.02	n.a.^b	n.a.^b	n.a.^b	n.a.^b
Per Brain Weight^a	%					n.a.^b	n.a.^b	n.a.^b	n.a.^b
LUNGS
Absolute Weight^a	g	1.306	1.302	1.515	1.537	1.051	1.127	1.138	1.338
Per Body Weight^a	%	3.91	4.05	4.39	4.91	4.93	5.52	5.59	6.46
Per Brain Weight^a	%
OVARIES		n.a.^b	n.a.^b	n.a.^b	n.a.^b	0.0921	0.0768	0.0943	0.0742
Absolute Weight^a	g	n.a.^b	n.a.^b	n.a.^b	n.a.^b	4.34	3.77	4.62	3.56
Per Body Weight^a	%	n.a.^b	n.a.^b	n.a.^b	n.a.^b
Per Brain Weight^a	%	n.a.^b	n.a.^b	n.a.^b	n.a.^b

Conclusions:

A NOAEL of 49.5 mg/m³ was identified for males and females, based on increased lung weight and microscopic changes of the lung.

Executive summary:

In a subacute inhalation toxicity study, a petroleum derived calcium salt was administered to 5 Sprague-Dawley rats/sex/concentration by whole body exposure at concentrations of 0, 49.5, 156 or 260 mg/m³ for 6 hours per day, 5 days/week for a total of 28 days.

Statistically significant, dose related increases in lung weights occurred in the mid and top dose groups, with corresponding increases in intraalveolar macrophages and hyperplasia/hypertrophy of bronchiole epithelium. The LOAEL is 156 mg/m³, based on effects in the lung. The NOAEL is 49.5 mg/m³.

This subchronic toxicity study in the rat is acceptable and satisfies the guideline requirement (OECD 412) for a subchronic inhalation study in the rat.

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

881.58 mg/m³

Study duration:

subacute

Species:

rat

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was not conducted to recognised guidelines, nor to GLP. The study report was unclear in places.

Qualifier:

no guideline followed

Deviations:

yes

Remarks:

only two treatment doeses were included (guidleine reccomends 3) Elizerabethan collars were used to prevent ingestion. The test site was not covered with a gauze patch

GLP compliance:

no

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: HARE Rabbits for Research
- Weight at study initiation: males, 1.6-2.8kg; females, 1.6-2.5kg
- Housing: Individually in stainless steel cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 10-24°C
- Humidity (%): 17-65%
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

Type of coverage:

open

Vehicle:

other: mineral oil

Details on exposure:

TEST SITE
- Area of exposure: 6.5 x 5 cm
- Surface area: 25%
- Time intervals for shavings or clipplings: Monday and Thursday of each week

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Wiping with paper towels
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2 ml/kg

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

6 hours

Frequency of treatment:

5 times per week

Remarks:

Doses / Concentrations:
0, 25, 100 %
Basis:
other: w/v in primol 205

No. of animals per sex per dose:

15

Control animals:

yes, concurrent vehicle

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

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
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Termination
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: Termination 10/sex/group

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: Yes
- How many animals:Termination: 10/sex/group

URINALYSIS: No
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / No data
- Animals fasted: Yes / No / No data
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

ANOVA with Dunnett's test
Regression analysis
Kruskal-Walis and Dunn's summed rank test
Joncheere's test for monotonic trend

Clinical signs:

effects observed, treatment-related

Dermal irritation:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
There were a number of mortalities in the study, One control and four high dose animals died or were sacrificed early in the study. One control female was sacrificed during the recovery phase. Two high dose males were sacrificed moribund during the treatment period. one high dose male was found dead during recovery. One high dose female was sacrificed during recovery. Cause of death was not established.

Alopecia was observed throughout the study in treated animals. In addition there were numerous incidences of erythema, oedema, atonia, desquamation, fissuring and exfoliation in treated animals but without dose response. These findings decreased in recovery phase.

BODY WEIGHT AND WEIGHT GAIN
Mean body weights were slightly reduced in treated groups but evaluation is confounded by the small number of animals.

FOOD CONSUMPTION
Not recorded

FOOD EFFICIENCY
Not recorded

WATER CONSUMPTION
Not recorded

OPHTHALMOSCOPIC EXAMINATION
Not recorded

HAEMATOLOGY
The mean total leukocyte count of low and high dose females was lower at termination of treatment. Mean haemaglobin and haemocrit values and mean erythrocyte counts were also reduced.

CLINICAL CHEMISTRY
Decreases in total protein and globulin levels occurred.

URINALYSIS
Not recorded

NEUROBEHAVIOUR
Not recorded

ORGAN WEIGHTS
Absolute and relative weights of testes and epididymis were decreased in low and high dose males, together with increases in mean and absolute liver weights.

GROSS PATHOLOGY
Discolouration of the liver was observed in males and females. Testes were small.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic examination revealed treatment related morphological changes in the skin, testes, epididymis and possibly liver.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
Not recorded

HISTORICAL CONTROL DATA (if applicable)
Not recorded

Dose descriptor:

NOAEL

Basis for effect level:

other: Effects were observed at the lowest dose tested, therefore a NOAEL cannot be identified from this study.

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Dose descriptor:

LOAEL

Effect level:

< 250 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: Effects observed at the lowest dose, therefore an arbitary LOAEL has been identified.

Critical effects observed:

not specified

Table 1: Incidence of selected pathologies

Parameter	n=10/sex	Dose level
Parameter	n=10/sex	Control	25	100	Recovery control	Recovery 25	Recovery 100
Small testis	M	0/10	3/10	6/13	0/5	0/5	1/3

Table 2: Absolute and relative organ weights

		Males				Females
DAILY DOSE (units e.g. mg/kg bw/day)		0	25	100	0		25	100
NUMBER OF ANIMALS		10	10	10	10		10	10
BODY WEIGHT (g)^a		-	-	-	-		-	-
LIVER
Absolute Weight^a	g	70.850	74.006	92.314	73.677		82.772	90.501
Per Body Weight^a	%	2.93	3.11	3.83	2.72		3.05	3.95
TESTES					n.a.^b		n.a.^b	n.a.^b
Absolute Weight^a	g	3.047	2.421	1.997	n.a.^b		n.a.^b	n.a.^b
Per Body Weight^a	%	12.49	10.16	7.76	n.a.^b		n.a.^b	n.a.^b

^aGroup means at the end of terminal necropsy are shown.

^bn.a. = not applicable

* p<0.05, ** p<0.01

Conclusions:

Toxicity occurred at all doses tested, therefore a NOAEL has not been identified. an arbitary LOAEL of 250 mg/kg bw has been identified, based on effects at the lowest dose.

Executive summary:

In a repeat-dose dermal toxicity study, an alkaryl magnesium salt derivative was applied to the shaved skin of 15white rabbits/sex/dose at dose levels of 0, 25 and 100%, 6 hours/day for 5 days/week during a 28-day period.

A NOAEL cannot be identified from this study, as effects were observed at the lowest dose.

This dermal toxicity study in the rabbit is acceptable.

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was not conducted to recognised guidelines, nor to GLP. The study report was unclear in places.

Qualifier:

no guideline followed

Deviations:

yes

Remarks:

only two treatment doeses were included (guidleine reccomends 3) Elizerabethan collars were used to prevent ingestion. The test site was not covered with a gauze patch

GLP compliance:

no

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: HARE Rabbits for Research
- Weight at study initiation: males, 1.6-2.8kg; females, 1.6-2.5kg
- Housing: Individually in stainless steel cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 10-24°C
- Humidity (%): 17-65%
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

Type of coverage:

open

Vehicle:

other: mineral oil

Details on exposure:

TEST SITE
- Area of exposure: 6.5 x 5 cm
- Surface area: 25%
- Time intervals for shavings or clipplings: Monday and Thursday of each week

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Wiping with paper towels
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2 ml/kg

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

6 hours

Frequency of treatment:

5 times per week

Remarks:

Doses / Concentrations:
0, 25, 100 %
Basis:
other: w/v in primol 205

No. of animals per sex per dose:

15

Control animals:

yes, concurrent vehicle

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

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
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Termination
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: Termination 10/sex/group

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: Yes
- How many animals:Termination: 10/sex/group

URINALYSIS: No
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / No data
- Animals fasted: Yes / No / No data
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

ANOVA with Dunnett's test
Regression analysis
Kruskal-Walis and Dunn's summed rank test
Joncheere's test for monotonic trend

Clinical signs:

effects observed, treatment-related

Dermal irritation:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
There were a number of mortalities in the study, One control and four high dose animals died or were sacrificed early in the study. One control female was sacrificed during the recovery phase. Two high dose males were sacrificed moribund during the treatment period. one high dose male was found dead during recovery. One high dose female was sacrificed during recovery. Cause of death was not established.

Alopecia was observed throughout the study in treated animals. In addition there were numerous incidences of erythema, oedema, atonia, desquamation, fissuring and exfoliation in treated animals but without dose response. These findings decreased in recovery phase.

BODY WEIGHT AND WEIGHT GAIN
Mean body weights were slightly reduced in treated groups but evaluation is confounded by the small number of animals.

FOOD CONSUMPTION
Not recorded

FOOD EFFICIENCY
Not recorded

WATER CONSUMPTION
Not recorded

OPHTHALMOSCOPIC EXAMINATION
Not recorded

HAEMATOLOGY
The mean total leukocyte count of low and high dose females was lower at termination of treatment. Mean haemaglobin and haemocrit values and mean erythrocyte counts were also reduced.

CLINICAL CHEMISTRY
Decreases in total protein and globulin levels occurred.

URINALYSIS
Not recorded

NEUROBEHAVIOUR
Not recorded

ORGAN WEIGHTS
Absolute and relative weights of testes and epididymis were decreased in low and high dose males, together with increases in mean and absolute liver weights.

GROSS PATHOLOGY
Discolouration of the liver was observed in males and females. Testes were small.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic examination revealed treatment related morphological changes in the skin, testes, epididymis and possibly liver.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
Not recorded

HISTORICAL CONTROL DATA (if applicable)
Not recorded

Dose descriptor:

NOAEL

Basis for effect level:

other: Effects were observed at the lowest dose tested, therefore a NOAEL cannot be identified from this study.

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Dose descriptor:

LOAEL

Effect level:

< 250 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: Effects observed at the lowest dose, therefore an arbitary LOAEL has been identified.

Critical effects observed:

not specified

Table 1: Incidence of selected pathologies

Parameter	n=10/sex	Dose level
Parameter	n=10/sex	Control	25	100	Recovery control	Recovery 25	Recovery 100
Small testis	M	0/10	3/10	6/13	0/5	0/5	1/3

Table 2: Absolute and relative organ weights

		Males				Females
DAILY DOSE (units e.g. mg/kg bw/day)		0	25	100	0		25	100
NUMBER OF ANIMALS		10	10	10	10		10	10
BODY WEIGHT (g)^a		-	-	-	-		-	-
LIVER
Absolute Weight^a	g	70.850	74.006	92.314	73.677		82.772	90.501
Per Body Weight^a	%	2.93	3.11	3.83	2.72		3.05	3.95
TESTES					n.a.^b		n.a.^b	n.a.^b
Absolute Weight^a	g	3.047	2.421	1.997	n.a.^b		n.a.^b	n.a.^b
Per Body Weight^a	%	12.49	10.16	7.76	n.a.^b		n.a.^b	n.a.^b

^aGroup means at the end of terminal necropsy are shown.

^bn.a. = not applicable

* p<0.05, ** p<0.01

Conclusions:

Toxicity occurred at all doses tested, therefore a NOAEL has not been identified. an arbitary LOAEL of 250 mg/kg bw has been identified, based on effects at the lowest dose.

Executive summary:

In a repeat-dose dermal toxicity study, an alkaryl magnesium salt derivative was applied to the shaved skin of 15white rabbits/sex/dose at dose levels of 0, 25 and 100%, 6 hours/day for 5 days/week during a 28-day period.

A NOAEL cannot be identified from this study, as effects were observed at the lowest dose.

This dermal toxicity study in the rabbit is acceptable.

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

5.13 mg/cm²

Species:

other: Human voluntary and laboratory species

The repeat dose toxicity has been determined by subacute 28-day oral toxicity studies by oral, dermal and

inhalation exposure. Based on the data available, the substance may have the potential for haemotological

effects with a reduction in cholesterol observed in one of the studies by oral exposure.

Effects observed in the repeat dose inhalation toxicity study available demonstrated enlarged lungs in high and

intermediate dose animals which are likely to be physical effects due to the inhalation of mineral oil [Test

material: Product as manufactured in mineral oil solvent further diluted in mineral oil (65/35)] and not

necessarily a direct toxicological effect of the registered substance. The data are therefore considered unsuitable

for determination of the intrinsic hazard of the substance as effects due to the registered substance may well be

masked by the effects due to inhalation of mineral oil mist. For example, Occupation Exposure Limits (OELs)

expressed for human exposure in the work place are typically 5 mg/m³. Considering a typical intraspecies

assessment factor of 10 the results demonstrate good correlation to the anticipated effect level for mineral oil

mist.

In view of this it is considered justifiable, for the purposes of assessing DNELs, to extrapolate inhalation hazard

from the oral exposure data available.

Oral-to-inhalation extrapolation was based on the following conditions:

(1) the available repeated dose toxicity study on EC 274-263-7 was reliable;

(2) the critical effect for the oral exposure were systemic, i.e., decreased serum cholesterol levels;

(3) the considered systemic toxic effects was independent of the route of exposure;

(4) the chemical is relatively soluble in body fluid.

RtR extrapolation may require corrections for difference in absorptions between oral (starting route) and the

inhalation (extrapolation route) (Dethloff, L. Z. 1993; Gerrity T. R., 1990; Sharrat M. 1988). Rennen et al. ran a

critical assessment on oral-to-inhalation route extrapolation for 215 substances with various physicochemical

properties. In this study, they compared the experimentally established NOAEL for inhalation study to the

values predicted from oral toxicity study by RtR extrapolation using various absorption assumptions, such as

100% oral and 100% inhalation; 100% oral and 75% inhalation; 50% oral and 100% inhalation. And they

demonstrated that when using systemic effect as criterion and under assuming equal absorption, 55% of the predication were regarded as “safe extrapolation”, whereas 45% as underestimation of the level of toxicity via

inhalation route. Based on this study, it is feasible to assume that equal absorption for this substance. Correcting

from oral to inhalation route of exposure was based on REACH guidance R8, and the mathematic formula was

described in DNEL derivations.

Furthermore, one dermal repeat dose study exists noting effects which resulted in the Study Director being

unable to assign a NOAEL. Due consideration of these data has been taken, but the results are considered not

representative of the toxicological effects of the substance since the observations have not been repeated in the

remaining study with dermal exposure, nor following exposure by oral and inhalation routes which are generally

considered to represent greater systemic exposure routes. Furthermore, effects to the testes of male rats were not

been observed in the reproductive toxicity study. In consequence, although data are not available to adequately

determine the cause of these effects, the fact that no similar observations have been found in any other study

undertaken is considered adequate justification to regard these data as not representative of the toxicological

profile of the registered substance.

Notwithstanding these effects, no further effects were observed in the studies and each study achieved a

NOAEL. The data are therefore considered adequate to assess the toxicological profile of the substance and for

the purposes of classification.

The following information is taken into account for any hazard / risk assessment:

The toxicity of the substance has been assessed by the repeated exposure over a period of 28-days by the three

routes of exposure, oral, dermal and inhalation. Effects observed by oral exposure demonstrate a reduction in

serum cholesterol at the highest tested dose. Furthermore, the results obtained by inhalation exposure are

considered unsuitable for determination of the intrinsic hazard of the substance by inhalation due to the high

proportion of mineral oil in the test sample [Test material: Product as manufactured in mineral oil solvent

further diluted in mineral oil (65/35) ].

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

A NOAEL of 500 mg/kg bw/day was identified in this study.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:

The test article elicted a range of effects in the lung; accumulation of intraalveolar macrophages, hyperplasia/hypertophy of the bronchiole epithelium. While these effects were also seen in control animals, there was a dose response in mid and high dose treated animals which was considered treatment related. A NOAEL of 49.5 mg/m³ was identified.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:

The test article elicted a range of effects in the lung; accumulation of intraalveolar macrophages, hyperplasia/hypertophy of the bronchiole epithelium. While these effects were also seen in control animals, there was a dose response in mid and high dose treated animals which was considered treatment related. A NOAEL of 49.5 mg/m³ was identified.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:

Toxicity occurred at all doses tested, therefore a NOAEL has not been identified. an arbitary LOAEL of 250 mg/kg bw has been identified, based on effects at the lowest dose.

Based on the NOAEL values available from these repeat dose toxicity data, the substance is considered to be not

classified under CLP for these repeat dose toxicity endpoints.