Naphthenic acids, reaction products with diethylenetriamine

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Rationale for read-across: naphthenic acid is presumed to be the toxic metabolite of NA-DETA Rationale for assigning reliability of 2: read-across, scientifically well performed study

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

2012

Reference 2

Reference Type:

publication

Title:

Naphthenic Acids Category Analysis and Hazard Characterization

Author:

The American Petroleum Institute

Year:

2012

Bibliographic source:

The American Petroleum Insititute

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)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Naphthenic acids

EC Number:

215-662-8

EC Name:

Naphthenic acids

Cas Number:

1338-24-5

Molecular formula:

"CnH2n+zO2, where n = carbon number and z = homologous group series number: z = 0 when no ring structures are present, z = -2 when 1 ring is present, z = -4 when 2 rings are present etc Result of high resolution mass spectroscopy of 4 naphthenic acids samples from different suppliers/sources: Ring distribution of aliphatic fraction: Z = 0 15-30% Z = -2 35-50% Z = -4 10-25% Z = -6 1-7% Z = -8 0-2% Z <= -6 1-5% (unsaturated compounds) Z >= -8 0-2% (unsaturated compounds) C<= 18 8-15% (N-containing compounds) C > 18 0-2% (N-containing compounds) Unknown 0-2% Distribution of carbon number (n) of 4 naphthenic acids samples from different suppliers/sources (n):C4-C27 (main: C8-C20)

IUPAC Name:

Naphthenic acids

Test material form:

other: oily liquids

Details on test material:

Blend of naphthenic acids from three sources.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

Male dosing was for 28-29 days
Females were dosed for 39-53 days

Frequency of treatment:

once per day

Doses / concentrations

Remarks:

Doses / Concentrations:
100, 300, 900 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Results and discussion

Effect levels

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Two high dose females were terminated on lactation day two; one was sacrificed in extremis due to acute inflammation of the uterus; the other was sacrificed due to total litter loss. All other rats survived to scheduled termination. Clinical observations, which were noted only in high dose group females and approximately an hour of dosing included hunched posture; rocking, lurching, and/or swaying while ambulating; walking on tiptoes; hypoactivity; and shallow respiration. Some of the high dose group males also exhibited hunched posture.

Body weight gain was reduced in high dose group males but the overall difference was less than 10% and the differences were not statistically significant. Among the females, the body weight gain in the high dose group was approximately 4% below control values but not significantly different at the end of the mating period. These differences in weight gain were associated with significantly reduced food consumption in the high dose group animals.

There were no statistically significant differences in parameters assessed as part of the functional observation battery including home cage observations, handling parameters, open field observations, sensory observations or neuromuscular observations. There were some small differences in body weight gain as indicated previously but other physiological parameters (catalepsy, body temperature) were not affected by treatment. There were also no differences in locomotor activity patterns (data not shown).

There were some hematology changes, primarily reductions in parameters related to hemoglobin content which were considered to have been treatment related. However, as is apparent from Table 1, the differences were small and there was no consistency between males and females.

The clinical chemistry values showed a similar pattern. All of the differences were small and within the historical range of the laboratory and there was no consistency of response between the sexes. In the absence of any corresponding pathological findings, these differences were most likely incidental.

The only notable gross observations were those of pale kidneys in the high dose males and a reduction in the number of corpora lutea in the high dose group females. Otherwise, the results of the gross examination were not remarkable. Organ weight determinations in males revealed significant increases in weights of liver, kidney, thyroid/parathyroid and epididymis although the differences in thyroid/parathyroid and epididymal weights were only statistically different when compared on a relative to body weight basis. In females, there was a significant increase in liver weights and significant reductions in lung weights, and absolute uterine weights (Table 2). The lung weights were within the historical range for the laboratory, and were not associated with any pathological changes. The uterine weights were not significantly different when compared relative to body weights. All gravid females were in lactational anaestrus and undergoing involution. Uterine weight values all fell within the historical range for the laboratory and were not associated with any gross, histopathologic or clinical pathology changes. Other than the uterine weights there were no microscopic differences in the reproductive organs of the male and female rats.

The results of the pathological investigation are summarized in Table 3. Kidney changes, reported in male rats only, were consistent with hyaline-droplet nephropathy (α2u-globulin-mediated nephropathy). The liver changes, found in organs from both male and female rats from the high dose group, were described as hepatocellular hypertrophy. Other changes included cortical lymphoid depletion of the thymus in females, primarily in rats from the high dose group. Epithelial hypertrophy and cytoplasmic vacuolation of the thyroid gland was noted in all treated animals, and cytoplasmic vacuolation of the zona fasciculate in the adrenal cortex was reported in males from all treatment groups and in high dose group females. The microscopic examination also revealed minimal cardiomyopathy which occurred with increased incidence in the males in the 100, 300 and 900 mg/kg/day groups, but the overall incidence was within the historical control range.

The gross and pathological assessments did reveal some differences that were treatment-related but were unlikely to have been toxicologically important. Liver weights were significantly increased in high dose groups of both male and female rats, and there was also a statistically significant increase in liver weight in the 300 mg/kg/day dose group in the males. The histological findings were essentially limited to minimal evidence of hepatocellular hypertrophy in the high dose group animals. As none of the liver enzyme markers were increased, this was most likely evidence of enhanced metabolic capacity and adaptive rather than adverse. Kidney weights were significantly elevated in the male rats from the high dose group, but not in the female rats. The histological evidence revealed the presence of hyaline droplets, mostly judged to have been of minimal severity, which increased in frequency in the male rats in a dose-dependent manner. As these were not found in female rats, the histological findings and gender-specificity, suggest the kidney changes were the consequence of an α-2u-globulin-related process which is male rat specific and not relevant to humans.

Minimal cardiomyopathy was reported to have increased in a dose-related fashion in male rats but was not considered to have been toxicologically important. In part because this is a common observation in control rats, and, additionally because the incidence was within the historical control range of the laboratory, the severity was not greater than that seen in the control groups, and because these microscopic observations were not associated with any other gross or clinical findings. Other changes included higher mean thyroid/parathyroid weights with corresponding epithelial hypertrophy and cytoplasmic vacuolation. The histologic changes were mostly judged as minimal. It is plausible that these changes reflected a compensatory response related to the increased metabolic capacity of the liver and more rapid turnover of thyroid hormones. Lymphoid depletion of the thymus was observed in the high dose females and microscopic findings of cytoplasmic vacuolation of the adrenal cortex were noted in the males and high dose group females. The lymphoid cortical depletion of the thymus and adrenal cortex vacuolation were considered to have been stress responses although cytoplasmic vacuolation of the adrenal cortex can also occur spontaneously or as the result of pharmacological effects . The overall no effect level for all systemic effects was 100 mg/kg/day.

Table 1. Results of Assessments of hematology and clinical chemistry parameters which were statistically different from control values.^1
Parameter Measured	Corn Oil Control	100 mg/kg/day	300 mg/kg/day	900 mg/kg/day
Males, data taken at terminal sacrifice
Red Blood Cell Count (10^6/ul)^2	9.22 ± 0.54	9.28 ± 0.28	8.91 ± 0.34	8.78 ± 0.22*
Hemoglobin (g/dL)^2	15.7 ± 0.72	15.8 ± 0.48	15.2 ± 0.54	14.7 ± 0.44*
Hematocrit (%)^2	48.1 ± 2.4	48.6 ± 1.6	46.5 ± 1.5	45.0 ± 1.8**
Platelet (10^3/ul)^2	854 ± 151	885 ± 84	803 ± 144	976 ± 87**
Leukocytes, absolute (10^3/ul)^2	0.02 ± 0.02	0.03 ± 0.02	0.02 ± 0.02	0.04 ± 0.03*
RDW (%)^2	11.4 ± 0.4	11.5 ± 0.4	11.6 ± 0.4	12.5 ± 0.6**
HDW (g/dL)^2	2.58 ± 0.10	2.68 ± 0.12	2.76 ± 0.16*	2.77 ± 0.27*
Females, data taken at termination (lactation day 4)
White blood cell count^2	5.15 ± 1.30	6.89 ± 1.58	7.68 ± 2.24*	7.59 ± 1.85*
APTT (seconds)^2	16.8 ± 1.9	15.9 ± 2.3	15.8 ± 3.1	13.9 ± 1.4 *
Lymphocytes, absolute (10^3/ul),	3.32 ± 0.61	4.50 ± 1.42	5.11 ± 1.75*	4.96 ± 1.60*
Monocytes, absolute (10^3/ul)	0.11 ± 0.10	0.24 ± 0.21	0.21 ± 0.12	0.35 ± 0.23*

 1. Parameters not affected by treatment included:

a. Males – white blood cell count, mean corpuscular volume (fL), mean corpuscular hemoglobin (pg), mean corpuscular hemoglobin content (g/dL), prothrombin time (sec), APTT (sec), reticulocytes (%), reticulocytes, absolute (10³/ul), MPV (fL), neutrophils (%), lymphocytes (%), monocytes (%), eosinophils (%), basophils (%), leucocytes(%), neutrophils, absolute (103/ul), lymphocytes, absolute (10³/ul), monocytes, absolute (10³/ul), eosinophils, absolute (10³/ul), basophils, absolute (10³/ul).

b. Females – red blood cell count (10⁶/ul), Hemoglobin content (g/dL), hematocrit (%), mean corpuscular volume (fL), mean corpuscular hemoglobin (pg), mean corpuscular hemoglobin content (g/dL), platelet count (10³/ul), prothrombin time (sec), reticulocytes (%), reticulocytes, absolute (103/ul), ), MPV (fL), neutrophils (%), lymphocytes (%), monocytes (%), eosinophils (%), basophils (%), leucocytes(%), neutrophils, absolute (10³/ul), eosinophils, absolute (10³/ul), basophils, absolute (10³/ul), Leukocytes absolute (10³/ul), RDW (%), HDW (g/dL)

 2. Data given as mean + SD

* = p < 0.05, ** = p < 0.01

 

Table 2. Statistically significant changes in terminal body weights and organ weights. The data are given as mean + SD.
Parameter	Sham Control	Corn Oil Control	100 mg/kg/day	300 mg/kg/day	900 mg/kg/day
Males
Final Body Weight	467 ± 27	454 ± 45	448 ± 45	439 ± 34	412 ± 28
Liver	15.61 ± 1.43	13.46 ± 2.01	13.98 ± 2.04	15.69 ± 1.83*	19.94 ± 2.08**
Kidney	3.51 ± 0.25	3.21 ± 0.20*	3.38 ± 0.39	3.53 ± 0.33	3.77 ± 0.46**
Heart	1.46 ± 0.09	1.46 ± 0.21	1.41 ± 0.14	1.43 ± 0.13	1.32 ± 0.13
Thyroid/parathyroid	0.019 ± 0.002	0.019 ± 0.001	0.020 ± 0.002	0.020 ± 0.002	0.020 ± 0.002
Epididymis (LT)	0.57 ± 0.14	0.60 ± 0.05	0.60 ± 0.04	0.66 ± 0.05*	0.63 ± 0.06
Epididymis (RT)	0.62 ± 0.04	0.62 ± 0.06	0.61 ± 0.03	0.66 ± 0.04	0.65 ± 0.06
Females
Final body Weight	335 ± 25	313 ± 23	301 ± 30	294 ± 24	289 ± 24
Liver	13.6 ± 2.0	11.7 ± 1.5	12.1 ± 1.1	13.3 ± 1.5	17.9 ± 2.4**
Kidney	2.39 ± 0.17	2.07 ± 0.18*	2.11 ± 0.15	2.05 ± 0.25	2.17 ± 0.19
Heart	1.21 ± 0.23	1.10 ± 0.10	1.08 ± 0.10	1.07 ± 0.11	1.01 ± 0.13
Lungs	1.36 ± 0.13	1.40 ± 0.13	1.26 ± 0.12*	1.20 +± 0.12**	1.20 ± 0.07**
Uterus/Vagina	1.07 ± 0.19	1.00 ± 0.14	0.86 ± 0.08*	0.88 ± 0.11*	0.85 ± 0.12*

* = p < 0.05, ** = p < 0.01

Table 3. Summary of microscopic findings from rats treated with refined naphthenic acids.
Doses, mg/kg/day	Males				Females
	Corn Oil	100	300	900	Corn Oil	100	300	900
N	12	12	12	12	9	12	10	10
Kidney
Hyaline Droplets	0	3	10**	11**	0	0	0	0
Minimal	0	3	9**	9**	0	0	0	0
Mild	0	0	1	2
Nephropathy	0	0	2	9**	0	0	0	0
Minimal	0	0	2	5*	0	0	0	0
Mild	0	0	0	4
Liver
Hypertrophy, hepatocellular, centrilobular	0	0	0	8**	0	0	0	10**
Minimal	0	0	0	8**	0	0	0	10**
Vacuolation, hepatocellular	2	1	2	0	0	1	0	2
Minimal	1	1	2	0	0	1	0	2
Mild	1	0	0	0	0	0	0	0
Thymus
Depletion, lymphoid, cortex	0	0	0	0	0	1	0	5
Minimal	0	0	0	0	0	1	0	4
Mild	0	0	0	0	0	0	0	1
Thyroid
Hypertrophy, epithelial	0	6	9*	11**	0	3	4	8**
Minimal	0	6	9**	11**	0	3	4	6*
Mild	0	0	0	0	0	0	0	2
Vacuolation, cytoplasmic	0	6	9**	10**	0	3	4	8**
Minimal	0	6	9**	10**	0	3	4	8**
Adrenal Cortex
Vacuolation, cytoplasmic	0	2	3	2	0	0	0	2
Minimal	0	2	3	2	0	0	0	1
Mild	0	0	0	0	0	0	0	1
Heart
Cardiomyopathy
Minimal	4	7	8*	8*	3	3	2	5
Mild	2	0	0	0	0	0	0	0

* = p < 0.05, ** = p < 0.01

Applicant's summary and conclusion

Conclusions:

The commercial naphthenic acid was investigated for its repeated dose toxicity according to the OECD Guideliine 422. The NOAEL was 100 mg/kg bw. The introduced study is proposed to be used to derive the subchronic toxicity of NA-DETA by read-across.

Executive summary:

The commercial naphthenic acid was investigated for its repeated dose toxicity according to the OECD Guideline 422. The applied doses were 0, 100, 300 and 900 mg/kg bw. All animals survived to scheduled termination except two females of 900 mg/kg bw group. The terminal body weight of males and females of 900 mg/kg bw were reduced. Reduced red blood cell count for males of 900 mg/kg bw and increased white blood cell count for females of 900 and 300 mg/kg bw were observed. Liver weight was increased for animals of 900 mg/kg bw and kidney weight was increased for males only in this group. The histopathological findings comprised hepatocellular hypertrophy, hyaline-droplet nephropathy, cortial lymphoid depletion in thymus, epithelial hypertrophy and cytoplasmic vacuolation of the thyroid gland and cytoplasmic vacuolation in the adrenal cortex. The NOAEL was 100 mg/kg bw.

The introduced study is proposed to be used to derive the subchronic toxicity of NA-DETA by read-across.