Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002-04-24 to 2002-11-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: - OECD guideline compliant - GLP compliant
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
as at 1995-July-27
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
as described in Commission Directive 96/54/EC
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3050 (28-Day Oral Toxicity in Rodents) as at 2000-July
Deviations:
no
Qualifier:
according to
Guideline:
other: Ministry of Health and Welfare (MHW) Guidelines 1986, Japanese Chemical Substances Control Law 1973 of the Ministry of International Trade and Industry (MITI) amended 1986
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): MXDA/SM ADDUCT
- Physical state: pale yellow viscous liquid
- Analytical purity: not reported
- Impurities (identity and concentrations): not reported
- Isomers composition: not reported
- Purity test date: not reported
- Lot/batch No.: PMS-02A
- Expiration date of the lot/batch: not reported
- Stability under test conditions: not reported
- Storage condition of test material: room temperature in the dark, until 12 April 2002, thereafter, room temperature, in the dark, under nitrogen

The composition of test material is not reported in this study report but is reported for the same batch of the test substance in the study report "MXDA/SM ADDUCT: DETERMINATION OF GENERAL PHYSICO-CHEMICAL PROPERTIES" by O'Connor, 2002 (see e.g. IUCLID section 4.2. Melting point):
Component 1: 54.9 %
Component 2: 4.7 %
Component 3: 36.4 %
Component 4: 4.0 %

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Sprague-Dawley Crl:CD ® (SD) IGS BR
- Source: Charles River (UK) Limited, Margate, Kent, UK
- Age at study initiation: five to six wks
- Weight at study initiation: males: 129 to 171 g, females: 122 to 159 g,
- Fasting period before study: no
- Housing: group housing of 5 animals per sex and per cage in polypropylene grid-floor cages suspended over trays lined with absorbent paper, environmental enrichment: wooden chew blocks (B & K Universal Ltd., Hull, UK)
- Diet (e.g. ad libitum): ad libitum, pelleted diet (5LF2 (Certified) Diet, PMT Nutrition International, Nottingham, UK)
- Water (e.g. ad libitum): ad libitum, mains drinking water
- Acclimation period: 7 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
On several occasions, the relative humidity and temperature were outside these ranges, however, this was not thought to have affected the purpose or integrity of the study.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Remarks:
BP
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- prepared at the appropriate concentrations as a suspension
- stability and homogeneity of the test material formulations determined by Safepharm Analytical Laborator
- prepared weekly and stored at approximately +4°C in the dark

VEHICLE
- Justification for use and choice of vehicle (if other than water): not reported, but arachis oil is a standard vehicle in rodent oral studies
- Concentration in vehicle: 2.5, 7.5 and 25 mg/mL
- Amount of vehicle (if gavage): 2 ml/kg bw
- Lot/batch no. (if required): not reported
- Purity: not reported
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Technique:
- gas chromatography (GC) using an external standard technique.

Sample preparation:
- test material formulations extracted with acetonitrile to give a final, theoretical test material concentration of approximately 0.1 mg/mL

Standards:
- standard solutions of test material prepared in acetonitrile at a nominal concentration of 0.1 mg/ml.

Procedure:
The standard and sample solutions analysed by GC using the following conditions:
- GC system: Agilent Technologies 5890 or 6890, incorporating autosampler and workstation
- Column: DB-5 (15 m x 0.53 mm id x 1.5 pm film),
- Oven temperature program: initial 200°C for 3 min, rate 15°C/min, final 300°C for 5 min
- Injection temperature: 300°C
- Flame ionisation detector temperature: 300°C
- Injection volume: 1 µL
- Retention time: 8 min

Homogeneity Determinations
- thorough mixing of test material formulations and sampling in triplicate from the top, middle and bottom of the container

Stability Determinations
- test material formulations sampled and analysed initially and then after storage at approximately +4°C in the dark for fourteen days

Verification of Test Material Formulation Concentrations
- test material formulations sampled and analysed within three days of preparation
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
5, 15 and 50 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: not reported
- Rationale for animal assignment (if not random): animals randomly allocated to treatment groups using random letter tables and determination of group mean bodyweights to ensure similarity between the treatment groups
- Rationale for selecting satellite groups: high dose group and vehicle control group
- Post-exposure recovery period in satellite groups: 14 d
- Section schedule rationale (if not random): after the last treatment
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: immediately before dosing and one and five hours after dosing during the working week, immediately before dosing and one hour after dosing at weekends, during the treatment-free period twice daily, morning and afternoon (once daily at weekends)
- Cage side observations: overt signs of toxicity, ill-health or behavioural change

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: days 7, 14, 21 and 25 all animals, observed for signs of functional/behavioural toxicity
- Detailed clinical observations:
Gait, Hyper/Hypothermia, Tremors, Skin colour, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behaviour, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation

BODY WEIGHT: Yes
- Time schedule for examinations: d 0 (the day before the start of treatment), d 7, 14, 21 and 28 and, in the case of recovery group animals, d 35 and 42 and at terminal kill.

FOOD CONSUMPTION: Yes
- weekly per cage


FOOD EFFICIENCY:
- No

WATER CONSUMPTION : Yes
- Time schedule for examinations: daily, visually per cage


OPHTHALMOSCOPIC EXAMINATION: No, except for checks for Exophthalmia in the detailed clinical observations

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the respective study end (28 d or 42 d (recovery group)), where necessary repeat samples obtained by cardiac puncture prior to necropsy on Day 29 and Day 43 respectively
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: all per group
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the respective study end (28 d or 42 d (recovery group)), where necessary repeat samples obtained by cardiac puncture prior to necropsy on Day 29 and Day 43 respectively
- Animals fasted: No
- How many animals: all per group
- Parameters checked in table 1 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: during wk 4 (non-recovery animals) or during wk 6 (recovery animals)
- Metabolism cages used for collection of urine: Yes, collected overnight, maintained under conditions of normal hydration during collection but without access to food
- Animals fasted: No
- Parameters checked in table 1 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during wk 4
- Dose groups that were examined: all
- Battery of functions tested:
• sensory activity: animals individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli: grasp response, touch escape, vocalisation, pupil reflex, toe pinch, startle reflex (ST1058 Startle Test Meter (Benwick Electronics)), tail pinch, blink reflex
• grip strength: forelimb and hindlimb, automated grip strength meter
• motor activity / other: 44 infra-red beam automated activity monitors, animals randomly allocated to the activity monitors, performed at approximately the same time each day, under similar laboratory condition, evaluation period: sixteen hours per animal
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 2), animals killed by intravenous overdose of sodium pentobarbitone followed by exsanguination at d 29 or d 43 (recovery animals)
HISTOPATHOLOGY: Yes (see table 2)

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
at 50 mg/kg bw, not fully reversible
Mortality:
mortality observed, treatment-related
Description (incidence):
at 50 mg/kg bw, not fully reversible
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
at 50 mg/kg bw , fully reversible
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
at 50 mg/kg bw , fully reversible
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
at 50 mg/kg bw, fully reversible
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
at 50 mg/kg bw, fully reversible
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
at 50 mg/kg bw, fully reversible
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
at 50 mg/kg bw, fully reversible
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
at 50 mg/kg bw, fully reversible
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
at 50 mg/kg bw, fully reversible
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
at 15 and 50 mg/kg bw, not fully reversible
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
- no fatalities during the study period
- clinical signs: At 50 mg/kg/d (males and females) increased salivation prior to and/or up to ten minutes after dosing from Day 6 onwards
starting during the third week of treatment: hunched posture, pilo-erection, tiptoe gait, waddling gait, increased inhumation, respiratory pattern changes and staining of the external body fur. Observations began to regress following cessation of treatment with only hunched posture reported in recovery 50 mg/kg/d animals by the end of the 14-day treatment-free period.

BODY WEIGHT AND WEIGHT GAIN
- 50 mg/kg, males: reduction in bodyweight throughout the study period, no recovery to normal during the recovery period
- 50 mg/kg, females: reduced bodyweight during week 4 of the treatment period, reversion to normal during the recovery period

FOOD CONSUMPTION
- 50 mg/kg, males: reduction in dietary intake throughout the study period, most pronounced during weak 3 and 4 and only reverted to normal during the last weak of the recovery period
- 50 mg/kg, females: slightly reduced dietary intake during weeks 3 and 4 of the treatment period, reversion to normal during the recovery period

FOOD EFFICIENCY
- 50 mg/kg, males: reduction in food efficiency throughout the study period, most pronounced during weak 3 and 4 and decreased still in week 5 of the recovery period
- 50 mg/kg, females: slightly reduced food efficiency during weeks 3 and 4 of the treatment period, reversion to normal during the recovery period

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
- no effects in any treatment group

OPHTHALMOSCOPIC EXAMINATION
- not conducted

HAEMATOLOGY
- 50 mg/kg, males: increased platelet count and erythrocyte count; all values reverted to normal during the recovery period
- 50 mg/kg, females: increased platelet count; all values reverted to normal during the recovery period

CLINICAL CHEMISTRY
- 50 mg/kg, males: increased AST, ALT, cholesterol, reduced plasma glucose, albumin and total protein concentration; all values except ALT reverted to normal during the recovery period
- 50 mg/kg, females: increased AST, ALT, cholesterol; all values except ALT reverted to normal during the recovery period

URINALYSIS
- 50 mg/kg, males: increased urine volume along with reduced specific gravity, increased hemoglobin concentration in urine; all values reverted to normal during the recovery period
- 50 mg/kg, females: increased hemoglobin concentration in urine; all values reverted to normal during the recovery period

NEUROBEHAVIOUR
- behavioural assessment: 50 mg/kg, males and females: hunched position and pilo-erection
- sensory activity: no treatment related effects in any dose group
- motor activity/grip strength: 50 mg/kg, males and females: reduction in overall motor activity

ORGAN WEIGHTS
- 50 mg/kg/d, males: increased relative liver, kidney and adrenal weights; all values reverted to normal during the recovery period
- 50 mg/kg, females: increased relative liver and kidney; only liver weights reverted to normal during the recovery period

GROSS PATHOLOGY
- no treatment related effects in any dose group

HISTOPATHOLOGY: NON-NEOPLASTIC
- Liver: At 50 mg/kg/ d(males and females) foamy vacuolation of hepatocytes, generalised hepatocyte enlargement, and vacuolar distension of scattered cells, single cell hepatocyte necrosis (females only), lower incidence of glycogen-type vacuolation(males only); all effects reverted to normal during the recovery period
- Spleen: At 50 mg/kg/d (males and females) lymphoid hyperplasia and vacuolar distension of scattered cells with associated apoptosis; all effects reverted to normal during the recovery period
- Adrenals: At 50 mg/kg/d (males and females) higher grades of severity of vacuolation of cortical cells; all effects reverted significantly during the recovery period and reverted to normal in females at the and of this period
- Heart: At 50 mg/kg/d (and quote from study report also "possibly at 15 mg/kg/d") in females greater incidence of myocarditis was seen in relation to treatment; all effects reverted to normal during the recovery period
- Urinary bladder: At 50 mg/kg/d (females) treatment-related hyperplasia of the transitional epithelium; all effects reverted to normal during the recovery period
- Small intestine: At 50 mg/kg/d (males and females) vacuolation of lamina propria cells in the duodenum, jejunum, and ileum related to treatment; all effects reverted significantly during the recovery period, just a few animals showing residual changes in the ileum. At 15 mg/kg bw/d (males and females vacuolation of lamina propria cells in the ileum in a few animals
- Lungs: At 50 mg/kg/d (males and females) foamy alveolar macrophages prevalent throughout the lung parenchyma; none of these effects reverted during the recovery period
- Mesenteric lymph node: At 50 mg/kg/d (males and females) foamy histiocytes related to treatment with the test material in the mesenteric lymph nodes; residual lesions characterised by accumulations of macrophages with amorphous eosinophilic cytoplasm following completion of the treatment-free period
- Ovaries: At 50 mg/kg foamy vacuolation of corpora luteal cells, the effects reverted only partially during the recovery period
- Skelettal muscle: At 50 mg/kg/d (males and females) muscle fibre degeneration and necrosis, and proliferation of sarcolemmal nuclei. At 15 mg/kg bw/d (males and females) slight muscle fibre degeneration and necrosis, and slight proliferation of sarcolemmal nuclei. Effects reverted significantly during the recovery period.

HISTOPATHOLOGY: NEOPLASTIC
- no effects in any dose group

HISTORICAL CONTROL DATA
- all above effects compared to both control groups and histroricl control data

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed at this level
Dose descriptor:
LOAEL
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: increased incidence of myocarditis in females, vacuolation of lamina propria in the ileum in both sexes and muscle fibre degeneration/necrosis in both sexes

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

- Table 1: Group Mean Weekly Bodyweights and Standard Deviations (SD)

Dose Level (mg/kg/day)

Number of Animals

 

Bodyweight (g) at Day

0

7

14

21

28

35#

42#

Males

0 (Control)

10

mean

155

223

286

348

367

427

450

 

 

sd

9

13

16

18

23

19

19

5

5

mean

149

216

279

334

336

-

-

 

 

sd

9

14

19

23

27

-

-

15

5

mean

149

213

273

327

334

-

-

 

 

sd

11

16

23

31

30

-

-

50

10

mean

150

209

260

288

281

310

336

 

 

sd

10

15

22

25

21

11

12

Females

0 (Control)

10

mean

137

169

195

214

223

249

246

 

 

sd

9

12

15

15

17

23

20

5

5

mean

139

175

197

219

227

-

-

 

 

sd

7

5

7

8

9

-

-

15

5

mean

133

166

194

214

222

-

-

 

 

sd

6

6

8

5

5

-

-

50

10

mean

135

167

185

196

197

217

223

 

 

sd

11

12

17

18

21

9

8

# = five animals per dose group during treatment-free period

- = not applicable

- Table 2: Group Mean Weekly Bodyweight Gains and Standard Deviations (SD)

Dose Level (mg/kg/day)

Number of Animals

 

Increase in Bodyweight (g) during Week

 

 

 

1

2

3

4

5#

6#

Males

0 (Control)

10

mean

68

64

61

19

44

23

sd

5

5

7

26

2

3

5

5

mean

67

63

56

2

-

-

sd

9

8

6

5

-

-

15

5

mean

64

60

54

7

-

-

sd

6

7

11

9

-

-

50

10

mean

3359

**51

***28

-7

***17

25

sd

7

9

11

25

7

3

Females

0 (Control)

10

mean

33

25

19

9

20

-2

sd

5

8

5

8

4

4

5

5

mean

37

21

22

8

-

-

sd

3

3

7

6

-

-

15

5

mean

33

28

20

8

-

-

sd

4

4

5

5

-

-

50

10

mean

31

18

12

*0

17

*5

sd

4

7

10

7

6

4

* = significantly different from corresponding control group p <0.05

*** = significantly different from corresponding control group p <0.001

# = five animals per dose group during treatment-free period

- = not applicable

Results in support of a LOAEL of 15 mg/kg bw/d:

- Table 3: Summary Incidence of Histopathological Findings

Histopathological Finding

Dose Level (mg/kg/day)

 

0 (Control)

5

15

50

0 (Control) Recovery group

50 Recovery group

Number of animals examined at terminal kill

5

5

5

5

5

5

MALES

Heart

Focal myocarditis

 

 

 

 

 

 

absent

1

1

1

0

2

2

(minimal)

3

4

4

3

3

3

(slight)

0

0

0

2

0

0

(moderate)

1

0

0

0

0

0

Duodenum

Vacuolation oflamina propria cells

 

 

 

 

 

 

absent

5

5

5

4

5

5

(minimal)

0

0

0

1

0

0

Jejunum

Vacuolation oflamina propria cells

 

 

 

 

 

 

absent

5

5

5

0

5

5

(minimal)

0

0

0

1

0

0

(slight)

0

0

0

2

0

0

(moderate)

0

0

0

2

0

0

Ileum

Vacuolation oflamina propria cells

 

 

 

 

 

 

absent

5

5

3

0

5

3

(minimal)

0

0

2

2

0

2

(slight)

0

0

0

3

0

0

Skelettal muscle

Fibre degeneration/necrosis

 

 

 

 

 

 

absent

4

5

5

0

4

3

(minimal)

1

0

0

0

1

2

(slight)

0

0

0

4

0

0

(moderate)

0

0

0

1

0

0

Proliferation sarcolemmal nuclei

 

 

 

 

 

 

absent

4

5

3

0

3

0

(minimal)

1

0

2

0

2

3

(slight)

0

0

0

1

0

1

(moderate)

0

0

0

3

0

1

(marked)

0

0

0

1

0

0

FEMALES

Heart

Focal myocarditis

 

 

 

 

 

 

absent

5

4

2

0

2

0

(minimal)

0

1

3

1

3

5

(slight)

0

0

0

4

0

0

Pericardial inflammation and abscess

 

 

 

 

 

 

absent

5

5

5

5

4

5

present

0

0

0

0

1

0

Duodenum

Vacuolation oflamina propria cells

 

 

 

 

 

 

absent

5

5

5

4

5

5

(minimal)

0

0

0

1

0

0

Jejunum

Vacuolation oflamina propria cells

 

 

 

 

 

 

absent

5

5

5

0

5

4

(minimal)

0

0

0

1

0

1

(slight)

0

0

0

3

0

0

(moderate

0

0

0

1

0

0

Ileum

Vacuolation oflamina propria cells

 

 

 

 

 

 

absent

5

5

2

2

5

2

(minimal)

0

0

3

2

0

0

(slight)

0

0

0

1

0

3

Skelettal muscle

Fibre degeneration/necrosis

 

 

 

 

 

 

absent

5

5

5

0

5

4

(minimal)

0

0

0

3

0

0

(slight)

0

0

0

2

0

1

Proliferation sarcolemmal nuclei

 

 

 

 

 

 

absent

5

5

3

0

4

2

(minimal)

0

0

2

1

1

2

(slight)

0

0

0

1

0

1

(moderate)

0

0

0

2

0

0

(marked)

0

0

0

1

0

0

Applicant's summary and conclusion

Conclusions:
Oral administration of the test material MXDA/SM Adduct, to rats for a period of twenty-eight consecutive days (according to OECD 407 and GLP) at dose levels of up to 50 mg/kg/day resulted in toxicologically significant effects at 50 and 15 mg/kg/day. However, the majority of effects in 50 mg/kg/day recovery group were reversible, whereas the effects occured in 15 mg/kg/day dose group were marginal and offten equivalent to findings in the control group.Therefore resulting LOAEL is proposed at 50 mg/kg bw/d, and the NOAEL at 15 mg/kg bw/d.
Executive summary:

Methodology

The study was designed to investigate the systemic toxicity of the test material and complies with the following regulatory guidelines:

OECD Guideline for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in the Rodent".

The test material was administered by gavage to three groups, each of five male and five rats, for twenty-eight consecutive days, at dose levels of 5, 15 and 50 mg/kg/day. A control group of five males and five females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of five males and five females, were treated with the high dose (50 mg/kg/day) or vehicle alone for twenty-eight consecutive days and then maintained without treatment for a further fourteen days.

Clinical signs, functional observations, bodyweight development, food and water consumption were monitored during the study. Haematology, blood chemistry and urinalysis were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment-free period. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results

There were no deaths during the study. No effects observed at all dose levels in endpoints water consumption and gross pathology.

For endpoints body weight / weight gain, food comsumtion, food efficiency, haematology, clinical chemistry, urinalysis, neurobehaviour, andorgan weights, effects were reported at the high dose level 50 mg/kg bw/d, however all effects fully reversible.

Concerning clinical signs, increased salivation prior to and/or up to ten minutes after dosing was detected at the 50 mg/kg/day dose level from Day 6 onwards. The physical condition of the animals began to deteriorate during the third week of treatment with the development of clinical signs including hunched posture, pilo-erection, tiptoe gait, waddling gait, increased inhumation, respiratory pattern changes and staining of the external body fur. Observations began to regress following cessation of treatment with only hunched posture reported in recovery 50 mg/kg/day animals by the end of the fourteen day treatment-free period. No such effects were detected at 15 or 5 mg/kg/day.

In histopathologic analysis several-related changes were observed at 50 mg/kg bw/d which all were fully reversible (foamy vacuolation of hepatocytes, generalised hepatocyte enlargement, and vacuolar distension of scattered cells, lymphoid hyperplasia and vacuolar distension of scattered cells with associated apoptosis, highher grades of severity of vacuolation of cortical cells,

hyperplasia of the transitional epithelium, foamy histiocytes in the mesenteric lymph nodes).

A greater incidence of myocarditis was seen in relation to treatment for females dosed at 50 mg/kg/day and possibly at 15 mg/kg/day. There was no toxicologically significant difference in the incidence or severity of myocarditis between recovery control and 50 mg/kg/day animals following completion of the recovery period.

Vacuolation of lamina propria cells in the duodenum, jejunum, and ileum was related to treatment for rats of either sex dosed at 50 mg/kg/day and for the ileum only in a few animals of either sex dosed at 15 mg/kg/day. Appreciable regression of the condition was observed for animals from the recovery 50 mg/kg/day treatment group, with just a few animals showing residual changes in the ileum.

Foamy alveolar macrophages were prevalent throughout the lung parenchyma in animals of either sex dosed at 50 mg/kg/day. There was no evidence of regression of the condition among recovery 50 mg/kg/day group animals following an additional fourteen days without treatment.

Foamy vacuolation of corpora luteal cells was seen in relation to treatment for females dosed at 50 mg/kg/day. Partial regression of the condition was observed for recovery 50 mg/kg/day females.

Muscle fibre degeneration and necrosis, and proliferation of sarcolernrnal nuclei were observed in relation to treatment for animals of either sex dosed at 50 mg/kg/day and to a much lesser extent for animals dosed at 15 mg/kg/day. Significant regression of both conditions was apparent for recovery 50 mg/kg/day animals following an additional fourteen days without treatment.

Conclusions

Finally it is concluded that oral administration of the test material MXDA/SM Adduct, to rats for a period of twenty-eight consecutive days at dose levels of up to 50 mg/kg/day resulted in toxicologically significant effects at 50 and 15 mg/kg/day. However, the majority of effects in 50 mg/kg/day recovery group were reversible, whereas the effects occured in 15 mg/kg/day dose group were marginal and offten equivalent to findings in the control group. No such effects were detected for animals of either sex at 5 mg/kg/day. Therefore resulting LOAEL is proposed at 50 mg/kg bw/d, and the NOAEL at 15 mg/kg bw/d.