Fatty acids, C14-18 and C16-18-unsatd., maleated, reaction products with oleylamine

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented and reported study fully adequate for assessment. The study was conducted according to an internationally accepted technical guideline and in compliance with GLP in a recognized contract research organization.

Cross-reference

Reason / purpose for cross-reference:

reference to same 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 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

of 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

liquid: viscous

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Sprague Dawley rats, strain: Crl:CD(SD) with appropriate range of bodyweight at study start.
- Source: Charles River (UK) Ltd.
- Age at treatment start: 72 days.
- Weight at treatment start: Males: minimum 337 g, maximum 417 g,
Females: minimum 227 g, maximum 293 g.
- Housing Inside a barriered rodent facility:
all animals pre-pairing + toxicity subgroups: In groups up to 5 by sex in solid floor polycarbonate cages.
during pairing (1 male+1 female/cage): In RB3 modified polycarbonate cages with stainless steel grid-floor over absorbent paper-lined trays.
males after pairing: In groups up to 5 in solid floor polycarbonate cages.
females during gestation and lactation: Females housed individually (+litter) in solid floor polycarbonate cages.
- Bedding material (in solid floor cages): Wood based bedding, sterilised by autoclaving before use.
- Cage enrichment: Aspen chew block + plastic shelter (except during pairing or post Gestation Day 20).
- Diet (ad libitum): Standard rodent diet (SDS VRF1 Certified) without antibiotic, chemotherapeutic or prophylactic agent.
- Fasting (diet withheld): Main phase males and Toxicity phase females overnight before blood sampling for clinical pathology.
- Water (ad libitum): Potable drinking water from the public supply.
- Acclimation period: 7 days before treatment start, under laboratory conditions.

Routine analysis of the batch of diet used and water, chew blocks and bedding material did not provide evidence of contamination that might have prejudiced the study.

IN-LIFE DATES:
- Duration of test, males & toxicity phase females: Five weeks
Duration of test, main phase females (i.e. reproductive subgroup): From 14 days prior to pairing to day 7 of lactation.
Duration of test, offspring: From birth to day 7 of lactation.

ENVIRONMENTAL CONDITIONS

Air conditioned room kept at positve pressure without re-circulation of the filtered fresh air supplied to the room.
Controlled environment, environmental conditions were set at:
- Temperature (°C): 21 ± 2°C
- Relative Humidity (%): 40 to 70%
- Photoperiod (artificial lighting): 12 hrs day / 12 hrs night
- Rate of air exchange: At least 15 changes/h
Deviations from the target ranges for temperature and relative humidity were not evident.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

- Concentration in vehicle: The concentration of the test material in vehicle varied between dose groups thus allowing constant dosage volume in terms of mL/kg bw/day.
- Amount (dose volume by gavage): 5 mL/kg bw/day.
Actual dose volumes were calculated at about weekly or shorter intervals accounting for the latest bodyweight. Litter animals were not dosed.

- For concentrations of test material in vehicle at different dose levels, see Table 1 in "Any other information on materials and methods incl. tables"

- Justification for choice of vehicle:
The suitability of corn oil as a vehicle was established during the 14-day range-finding study:
Endpoint study record "7.5.1 Repeated dose toxicity: oral - 14d_range-finding_gavage_HLS_GAH0175".
In addition, in the present main study, concentrations of dose formulations were chemically analysed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Chemical analysis of test material formulations by high performance liquid chromatography coupled with a mass spectrometer (HPLC-MS/MS).
- Mean concentrations of the test material formulations (verified for first and 7th treatment week) were confirmed at each dose level.
Chemical analysis confirmed that the mean concentrations of WS400107 in prepared formulations were within 93% to 100% of the corresponding
nominal concentration, thus confirming acceptable accuracy of formulation for dosing of the animals.
- Homogeneity and stability of test material formulations at 2 and 200 mg/L and at storage and handling conditions similar to those adopted for dosing of the animals were confirmed.

Duration of treatment / exposure:

- Treatment period, males & toxicity phase females: Daily, for five consecutive weeks, in males commencing 14 days prior to pairing
- Treatment period, main phase females (i.e. reproductive subgroup): 44 to 51 days (from 14 days prior to pairing to Day 6 of lactation)
- Offspring were not dosed

Frequency of treatment:

Daily, 7 days/week (during parturition, dosing omitted as appropriate)

Doses / concentrations

Remarks:

Doses / Concentrations:
0 (vehicle control), 150, 400, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

Toxicity phase animals: */ 5 females
Main phase animals (i.e. reproductive subgroups): 10 males / 10 females
*Explanatory note by the notifier:
Examinations assigned to the toxicity phase females to meet the requirements of a 28-day repeat dose oral toxicity study were also assigned to 5 (for some examinations to 10) main phase males per dose group. Therefore, these 5 main phase males per dose group are called also "toxicity subgroup" in the present robust study summary for clarification. After pairing with main phase females, all males (F0) were killed at the same time (Week 6).

Control animals:

yes, concurrent vehicle

Details on study design:

This study was conducted to examine both repeated dose toxicity and  reproductive/developmental toxicity as an OECD screening combined study
(OECD 422 test guideline).  Therefore, animals initially entering the study were divided into toxicity subgroup animals (toxicity phase) and reproductive subgroup animals (main phase), whereby 5 of the 10 F0 males (used for pairing) per dose group formed the toxicity male subgroups.

Dose selection was based on the results of a 14-day preliminary oral (gavage) toxicity study in the rat in which dose levels of 100, 300 or 1000 mg/kg/day did not have any severe toxic effects on young adult animals (females nulliparous and non-pregnant) preventing the choice of up to 1000 mg/kg/day in the present OECD 422 toxicity study.

Positive control:

Not included in the study.

Examinations

Observations and examinations performed and frequency:

Clinical observations performed and frequency:
- Clinical signs : At least twice a day
- Detailed physical examination
and arena observations: Before treatment start and at least once a treatment week (except possibly 1 female taking 5 to 8 days for pairing).
- Functional Observation Battery:* During treatment week 5 (before dosing) on all toxicity subgroup animals (5 males + 5 females/group).
- Body weight, all males: About weekly throughout the study.
Body weight, Toxicity Females: About weekly throughout the study.
Body weight, Repro. Females: Weekly for pre-pairing period; on gestation days 0, 6, 13, 20; on lactation days 1, 4 & 7.
- Food consumption, all males: Weekly for pre-pairing period and for the period after mating.
Food cons., Toxicity Females: About weekly throughout the study.
Food cons., Repro. Females: Weekly for pre-pairing period, during gestation for days 0-6, 6-13, 13-20, during lactation for days 1-4 & 4-7.

* FOB including sensory reactivity tests (approach, touch, auditory startle reflex, tail pinching), grip strength and motor activity.

Haematological examinations (only for toxicity subgroup animals) during treatment week 5 after functional observation battery:
Red blood cell count, reticulocyte count, white blood cell count, platelet count, hemoglobin concentration, hematocrit value, differential leukocyte counts,
protrombin time, activated partial thromboplastin time, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration.

Blood (plasma) chemical examinations (only for toxicity subgroup animals) during treatment week 5 after functional observation battery:
Total protein, albumin, A/G ratio, urea, creatinine, glucose, total cholesterol, triglycerides, total bilirubin, bile acids, sodium, potassium, chloride,
calcium, inorganic phosphorus, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase.

This study was conducted to examine both repeated dose toxicity and  reproductive/developmental toxicity as an OECD screening combined study
(OECD 422 test guideline).  Therefore, some of the examinations were confined to toxicity subgroup animals, as indicated above.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, see below
WEIGHING OF ORGANS: Yes, see below
HISTOPATHOLOGY: Yes, see below

Terminal sacrifice
- all males (F0) and toxicity subgr. females: Killed in Week 6, after completion of the Treatment Week 5 investigations.
- reproductive subgr. females & offspring: Killed on Day 7 post partum.
(1 high dose female was killed after mating in poor clinical condition. It was confirmed to be non-pregnant)
(1 control female failed to mate and therefore was killed in Week 8 following 7 weeks of treatment)
Gross pathology:
- adult/parental animals: Full macroscopic examination.
- offspring, Lactation Day 7: Careful external macroscopic examination of all survivors for gross abnormalities.
- externally abnormal offspring
and premature deaths: Internal macroscopic examination including assessment of the presence of milk in the stomach, where possible. (Missing or grossly autolysed or cannibalised offspring could not be examined).
Organs Weights:
- all males (F0) +
toxicity subgroup females: Adrenals, brain, epididymides, heart, kidneys, liver, ovaries, prostate, seminal vesicles with coagulation gland,
spleen, testes, thymus, uterus with cervix & oviducts.
- dams: Ovaries

Histopathology:
- toxicity subgroups: The following organs were microscopically observed for the control and 1000 mg/kg bw/day toxicity subgroups:
Brain, eyes, pituitary gland, thyroid with parathyroids, heart, thymus, liver, spleen, adrenals, kidneys, testes,
epididymides, ovaries, lung, trachea, oesophagus, stomach, duodenum, jejunum, ileum, caecum, rectum, colon,
Peyer's patch, lymph node (axillary, mesenteric), urinary bladder, uterus (with cervix & oviducts), vagina,
spinal cord, sciatic nerve, skeletal muscle, sternum with marrow, seminal vesicle & coagulation gland, prostate.
In addition, the duodenum, jejunum, ileum, mesenteric lymph nodes, seminal vesicles and prostate
were also examined by light microscopy for the remaining F0 males of the control and 1000 mg/kg bw/day
groups and all adult males and toxicity subgroup females of the 150 and 400 mg/kg bw/day groups and any
gross lesions for all adult animals.

- reproductive subgroups All above organs/tissues from a premature death (1 high dose female). Gross lesions from adult dams comprised only focal hairloss not necessating any examination by light microscopy.

Other examinations:

Reproductive and developmental toxicity parameters (addressed in separate endpoints).

Statistics:

Statistical analysis of grip strength, motor activity, bodyweight, food consumption, organ weight, litter size, survival indices & clinical pathology data:

- Parametric analysis, if Bartlett's test for variance homogeneity was not significant at the 1% level.
F1 approximate test for monotonicity of dose-response. If this F1 test was not significant at the 1% level, Williams' test for a monotonic trend was applied.
If this F1 test was significant, suggesting that the dose-response was not monotone , the Dunnett's test was performed instead.

- Non-parametric analysis, if Bartlett's test was still significant at the 1% level following logarithmic and square-root transformations.
H1 approximate test for monotonicity of dose-response. If this H1 test was not significant at the 1% level, Shirley's test for a monotonic trend was applied.
If this H1 test significant, i.e. dose-response not monotone, then Steel's test instead.

Grip strength, motor activity, survival indices and clinical pathology data
if 75% of the data (across all groups) were the same value, pairwise comparison of each dose group against the control by Fisher’s Exact tests.

Organ weight data
Covariance analysis using terminal bodyweight as covariate (Angervall & Carlstrom, 1963)

Gestation length
Exact (or asymptotic) two-tailed Linear-by-linear test (Cytel 1995), with equally spaced scores.*

Oestrous cycles
Exact (or asymptotic) 1-tailed (upper tail) Linear-by-linear test (Cytel 1995)*

* If significant (p<0.05), exclusion of the highest dose group and re-testing until no longer significant (p≥0.05).

For statistical references, see next field.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

attributable to treatment with the test material

Mortality:

no mortality observed

Description (incidence):

attributable to treatment with the test material

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

but not considered to represent adverse effects

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

but not considered to represent adverse effects

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

but toxicological significance is unclear

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

but not representing toxicity or toxicological significance unclear

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

but not representing toxicity or toxicological significance unclear

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS, NEUROBEHAVIOUR AND MORTALITY
Mortality, clinical signs or effects on sensory reactivity, grip strength or motor activity attributable to treatment with the test material were not evident. One high dose female of the reproductive subgroup was killed during the gestation phase, because of its poor clinical condition (thin built, hunched posture, piloerection, ungroomed fur and 13 g bodyweight loss over 14 days, confirmed to be not pregnant at necropsy). One control female of the reproductive subgroup failed to mate and therefore was killed after 7 weeks of treatment (Week 8).

BODYWEIGHT GAIN AND FOOD CONSUMPTION
A dose related adverse effect on bodyweight gain was evident in males over the 5 weeks of treatment attaining statistical significance at 400 and 1000 mg/kg bw/day. Nulliparous females were unaffected by treatment, but in dams treated with 1000 mg/kg bw/day bodyweight gain was statistically significantly lower than in concurrent controls during gestation Days 6 to 13 and overall during gestation Days 0 to 20. Food consumption was lower than in concurrent controls in males at 1000 mg/kg/day during the 5-week treatment period and in dams at 1000 mg/kg/day on lactation Days 4 to 7.

CLINICAL PATHOLOGY

Changes in haematology or clinical chemistry (blood plasma) parameters were not considered to represent toxic effects, because there were no histopathological correlates indicative of toxicity.

After 5 weeks of treatment at 400 or 1000 mg/kg/day, counts of total white blood cells, neutrophils or monocytes were statistically significantly higher than in concurrent controls in both sexes and basophil counts in females. In addition, prothrombin clotting time was statistically significantly higher than in concurrent controls in males treated at 1000 mg/kg/day and females at 400 or 1000 mg/kg/day. Mean corpuscular volume was marginally lower than in concurrent controls in males at 1000 mg/kg/day.

Alanine aminotransferase and aspartate aminotransferase were statistically significantly higher than in concurrent controls in both sexes at 400 and 1000 mg/kg/day, and urea in males at 400 mg/kg/day and both sexes at 1000 mg/kg/day. In addition albumin to globulin ratio was lower than in concurrent controls in both sexes at 1000 mg/kg/day. Females given 1000 mg/kg/day also had low levels of total protein, albumin, total bilirubin and calcium, and high levels of phosphorus and bile acids all of which attaining statistical significance when compared to concurrent controls.

ORGAN WEIGHTS
After 5 weeks of treatment at 1000 mg/kg/day, prostate and seminal vesicle weights were statistically significantly lower than in concurrent controls. The toxicological significance of this finding remained unclear. Any other organ weights or organ weights adjusted to terminal bodyweight statistically significantly different from concurrent controls did not have any histopathological correlates and were not considered to be indicative of toxicity.

GROSS PATHOLOGY AND HISTOPATHOLOGY (NON-NEOPLASTIC)
The effects on prostate and seminal vesicle weights were consistent with small prostates and small seminal vesicles macroscopically apparent and reduced colloid microscopically seen in these tissues in four of ten high dose males. The toxicological significance of this finding remained unclear.

Thickening of the jejunum, ileum and duodenum was macroscopically apparent in a number of animals from all test item treated groups and thickening of the colon in two of ten males of the high dose group (1000 mg/kg/day) after 5 weeks of treatment, and were attributed to treatment with the test material. Histopathology revealed foamy macrophages within the villi of the illeum and jejunum (at 150, 400 and 1000 mg/kg/day) and of the duodenum (at 400 and 1000 mg/kg/day). Macrophage aggregates in the mesenteric lymph nodes in all treated groups were considered to be secondary deposits of the foamy macrophages seen in the intestines. These pathology findings were considered not to represent toxicity, but to result from test material ingestion by macrophages.

An increased incidence of aggregations of alveolar macrophages in the lungs in both sexes at 1000 mg/kg/day has been of uncertain relationship to treatment. As this is a commonly observed background finding, it is probably fortuitous.

OTHER RESULTS
Reproductive and developmental toxicity parameters are addressed in separate endpoints.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

> 150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Reduced bodweight gain at 400 and 1000 mg/kg bw/day.

2

Dose descriptor:

NOAEL

Effect level:

> 400 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Bodweight gain in dams at 1000 mg/kg bw/day significantly lower than in concurrent controls during gestation, whilst unaffected in nulliparous females during the first five weeks of treatment.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

 

Table 2: Bodyweight and Bodyweight Change - Group Mean Values (g) for Main and Toxicity Phase Animals – Males
Group					1			2				3				4
Compound					Vehicle Control			WS400107				WS400107				WS400107
Dose (mg/kg/day)					0			150				400				1000
Group/Sex				Week	Week	Week	Week			Week	Week		Change	Change	Change		Change	Change	Change
				0	1	2	3			4	5		0-1	1-2	2-3		3-4	4-5	0-5
Statistical test:				Wi	Wi	Wi	Wi			Wi	Wi			Wi				Wi	Wi
1/M	Mean			371	408	439	455			480	497		37	31	16		25	17	125
	SD			18.9	22.9	30.9	30.3			34.0	39.3		7.2	9.0	10.8		8.1	7.6	26.1
	N			10	10	10	10			10	10		10	10	10		10	10	10
2/M	Mean			365	396	427	440			462	475		31	31	14		21	13	110
	SD			15.3	18.8	24.7	28.3			30.7	32.0		6.1	8.1	8.9		7.5	7.1	19.6
	N			10	10	10	10			10	10		10	10	10		10	10	10
3/M	Mean			376	405	431	441			463	465		29*	25	11		22	3**	89**
	SD			14.8	16.8	20.6	21.5			22.3	25.4		6.8	6.7	13.8		10.5	9.3	18.1
	N			10	10	10	10			10	10		10	10	10		10	10	10
4/M	Mean			369	382*	389**	396**			402**	400**		13**	7**	6		6**	-2**	31**
	SD			24.4	26.7	27.4	36.9			37.9	39.8		4.9	9.5	14.7		12.3	12.7	29.7
	N			10	10	10	10			10	10		10	10	10		10	10	10

 

Statistical test Wi = Treated groups compared to Control using Williams’ test,    * p<0.05,    ** p<0.01  

Table 3: Bodyweight and Bodyweight Change - Group Mean Values (g) for Main and Toxicity Phase Animals – Females
Group			1		2			3		4
Compound			Vehicle Control		WS400107			WS400107		WS400107
Dose (mg/kg/day)			0		150			400		1000
Group/Sex		Week	Week	Week	Week		Week	Week		Change	Change	Change
		0	1	2	3‡		4‡	5‡		0-2	2-5‡	0-5‡
Statistical test:		Wi	Wi	Wi	Wi		Wi	Wi		Sh	Wi	Wi
1/F	Mean	252	265	276	301		306	312		24	24	55
	SD	13.6	15.7	23.1	37.0		38.8	41.8		14.0	17.5	33.1
	N	15	15	15	6$		6$	6$		15	6$	6$
2/F	Mean	252	259	269	291		296	300		17	20	39
	SD	14.4	13.6	13.0	18.0		19.7	20.1		6.5	6.6	12.5
	N	15	15	15	5		5	5		15	5	5
3/F	Mean	257	265	273	280		283	285		16	13	31
	SD	12.5	12.7	13.8	18.6		18.1	17.2		8.5	4.4	12.9
	N	15	15	15	5		5	5		15	5	5
4/F	Mean	250	263	273	293		302	307		23	19	52
	SD	13.2	14.3	15.7	7.2		12.9	14.6		15.8	5.8	12.9
	N	15	15	15	6^		5	5		15	5	5

 

‡ Toxicity phase animals only

Statistical test Wi = Treated groups compared to Control using Williams’ test,    * p<0.05,    ** p<0.01

Statistical test Sh Treated groups compared to Control using Shirley’s test,    * p<0.05,    ** p<0.01

$ One Group 1 female (1F, 65) failed to mate and therefore remained in the treatment phase

^ One Group 4 female (4F, 43) was still in pairing during Week 3

 

Table 4: Bodyweight and Bodyweight Change - Group Mean Values (g) for Main Phase Females during Gestation
Group			1		2		3			4
Compound			Vehicle Control		WS400107		WS400107			WS400107
Dose (mg/kg/day)			0		150		400			1000
Group/Sex		Day	Day	Day	Day			Change	Change		Change	Change
		0	6	13	20			0-6	6-13		13-20	0-20
Statistical test:		Wi	Wi	Wi	Wi				Wi		Wi	Wi
1/F	Mean	274	300	337	431			26	37		94	157
	SD	13.2	14.3	12.1	22.1			8.1	6.4		12.7	13.4
	N	9	9	9	9			9	9		9	9
2/F	Mean	269	295	325	412			26	30		87	142
	SD	7.9	8.8	12.1	20.4			7.0	7.2		15.2	21.2
	N	10	10	10	10			10	10		10	10
3/F	Mean	272	299	330	418			27	31		88	146
	SD	11.7	11.3	14.0	20.8			7.2	9.5		11.1	15.7
	N	10	10	10	10			10	10		10	10
4/F	Mean	270	295	320**	396**			25	25**		76	126*
	SD	10.5	11.1	11.1	39.5			7.2	8.7		30.3	37.7
	N	9	9	9	9			9	9		9	9

 

Statistical test Wi = Treated groups compared to Control using Williams’ test,    * p<0.05,    ** p<0.01

 

Table 5: Bodyweight and Bodyweight Change - Group Mean Values (g) for Main Phase Females during Lactation
Group			1		2	3	4
Compound			Vehicle Control		WS400107	WS400107	WS400107
Dose (mg/kg/day)			0		150	400	1000
Group/Sex		Day	Day	Day			Change
		1	4	7			1-7
Statistical test:		Wi	Wi	Wi			Wi
1/F	Mean	334	340	357			23
	SD	15.1	21.4	11.8			9.9
	N	9	9	9			9
2/F	Mean	321	325	348			27
	SD	12.3	18.9	18.5			15.7
	N	10	10	10			10
3/F	Mean	312**	328	344			32
	SD	12.3	11.6	14.0			13.4
	N	10	10	10			10
4/F	Mean	307**	316**	330**			23
	SD	20.4	17.8	16.3			12.5
	N	9	9	9			9

 

Statistical test Wi = Treated groups compared to Control using Williams’ test,    * p<0.05,    ** p<0.01

Applicant's summary and conclusion

Conclusions:

The NOAELs of 150 mg/kg bw/day for male and 400 mg/kg bw/day for female rats were derived from bodyweight gain statistically significantly lower than in concurrent controls in males after 5 weeks of treatment at 400 and 1000 mg/kg bw/day and in dams during gestation at 1000 mg/kg bw/day. These findings were considered to represent adverse effects indicative of toxicity.

Microscopically visible foamy macrophages in the villi of the ileum, jejunum and duodenum and macrophage aggregates in the mesenteric lymph nodes were considered not to reflect a toxic effect of treatment, but to result from ingestion of the test material by macrophages. Possibly the presence of the foamy macrophages in the villi inhibited food consumption and/or nutrient absorption and thus caused reduced bodyweight gain.