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Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The following summary and discussion is to explain by weight of evidence why further investigations into fertility and pre- and postnatal development can be waived:

Background

ECHA performed a compliance check for naphthalene, which resulted in a request for performance of a 2-generation reproduction study on naphthalene. ECHA explicitly criticised that '… the Registrant has not demonstrated how, e.g. the 10 week pre-mating period, the test animal group size of not less than 20 pregnant females, the post-natal evaluation of the F1 generation and breeding and evaluation of the F2 generation information requirements could be covered by data from the available carcinogenicity and repeated dose toxicity studies.'

 

According to ECHA´s practical guide 'How to report weight of evidence', 'the way the weight of evidence is implemented is case-dependent. It is influenced by the relation between the amount of information needed and the importance of the decision to be taken, as well as by the likelihood and consequences of the decision based on that information being wrong.'

Therefore, it is the registrant´s understanding that the concept of weight of evidence is not solely limited to providing information identical to what would be available from a performed 2-generation reproduction toxicity study, in other words that sufficient weight of evidence is not only given in case all key information potentially available from this study can be well represented by equivalent alternatives. 

On the contrary, we think that the information and deliberations provided below are more comprehensive, various aspects going beyond the observations obtainablefrom a generation study, solely limited to the very animal species under test and the test-specific endpoints.

 

Available toxicological information

Naphthalene has been evaluated for toxicity in sub-chronic and chronic / oncogenicity studies in rat and mouse by various routes of exposure as well as in three intra-uterine developmental studies in rat and rabbit (see relevant IUCLID endpoint entries). In no case has been observed any effect in reproductive organs. Naphthalene toxicity to reproductive organs in rats was not observed at oral doses as high as 400 mg/kg bw/d in a 90-d repeated-dose study. Metabolism of naphthalene has provided no evidence for naphthalene itself or naphthalene metabolites to accumulate in the reproductive organs to a critical extent (see below). Since no changes in reproductive organs were observed, it is unlikely that fertility effects would be manifest. Regarding sperm parameters, testis and epididymal weight showed no alteration upon exposure to naphthalene in the previously mentioned studies. According to Hayes (Hayes, A. Wallace: Principles and Methods of Toxicology, Taylor and Francis, 2001, Fourth edition, page 1283), these parameters are better indicators for predicting effects on male fertility than sperm production rate, sperm count/g epididymis or percent motile sperm. Thus, it can be concluded that missing information on the latter three sperm parameters is of less importance.

Although naphthalene is known to cross the placental barrier, no developmental toxicity was observed in either rats and rabbits (see relevant IUCLID endpoint entries). Thus, although the foetus was exposed to naphthalene, no effects on foetal development were observed. No foetal growth effects were observed in rats at oral doses up to 450 mg/kg bw/d (in rabbits at 500 mg/kg bw/d), a dose that led to significant maternal toxicity in the rat dam. No implantation loss was observed indicating there was no effect on foetal viability. It can be concluded that developmental toxicity studies demonstrated that maternal toxicity occurs at lower doses than foetal toxicity.

 

There is no evidence to suggest that the juvenile animal is more susceptible to naphthalene than the young animals evaluated in the available long-term toxicity studies, which were initially dosed when the animals were approximately 6 weeks old. A two-generation reprotoxicity study is supposed to show if increased vulnerability of subsequent generations towards a substance can be observed. According to Hayes (Hayes, A.Wallace: Principles and Methods of Toxicology, Taylor and Francis, 2001, Fourth edition, page 1268), reproductive toxicity occurring at the parent generation, adverse effects occurring at sensitive developmental periods and the bioaccumulation potential of the test agent (naphthalene in this case) can help to make qualitative predictions to filial generations. Naphthalene has no bioaccumulation potential, and the cited studies above give evidence that neither reproductive nor developmental toxicity occur when naphthalene is administered in animal studies at high concentrations. Thus, the risk for reprotoxic effects on filial generations related to exposure to naphthalene is assumed to be low.

 

Metabolism of naphthalene

Naphthalene toxicity is associated with its metabolism. The primary step is epoxidation of the aromatic ring. The reactive epoxide, an electrophilic structure, may react with glutathione catalysed by glutathione S-transferases, but other detoxifying reactions are possible, mainly conjugation with glucuronic acid and/or sulphate.

In rodents, naphthalene is a glutathione-depleting agent in various organs: There is a good correlation between potential indicators for naphthalene toxicity and the GHS status in affected rodent tissues:

The study by Warren et al. (1982) shows the close dose-response relationship between naphthalene dosage and covalent binding of reactive intermediates to cellular macromolecules exemplified in liver, lung and kidney of mice. There is a distinct threshold for this marker at a dose of about 200 mg/kg bw (i.p.) in mice, which correlates with a significant depletion of the GSH pool (see entry Toxicokinetics).

The study of Gandy et al. (1990) describes the hepatic, testicular and epididymal GSH profile over time in male rats after a high single i.p. dose of 500 mg/kg bw. There is a dramatic decrease in GSH in the liver, - however, only very mild and transient in the testes and more marked in the epididymides. The level in the latter organ drops to about 50 % of normal while slowly recovering to 60 % without reaching the control level within the observation time of 16 h (see entry Toxicokinetics). (Note: Remarkably, despite the high hepatic GSH loss, the liver is not the target organ of naphthalene toxicity!)

This provides evidence that a high dose of naphthalene may partially deplete the GSH pool in the reproductive system of male rats, but is unlikely to induce adverse effects in the gonads, as the residual GSH pool is supposed to be sufficient to prevent reactive naphthalene intermediates from interaction with vital cellular structures, at least at the lower doses that can be achieved by the relevant route of inhalation.

 

·        Inhalation exposure

A model calculation for long-term inhalation exposure may show that there is little concern over an intoxication at relevant exposure concentrations:

 

·        Exposure concentration: 25 mg/m3(= ~5 ppm)

·        Absorption: 100 %

·        Duration: 8 h/d

·        Rat respiration volume: 20.5 L/h (default value: see Guidance R8, Table R.8-17)

·        Rat body weight: 0.5 kg (default value: see Guidance R8, Table R.8-17)

·        Weight-specific respiration volume: 0.0205 (m3/h) *8 h/ 0.5 kg = 0.328 m3/kg bw [per 8 h]

·        Dose: 25 (mg/m3) *0.328 m3/kg bw = ~8 mg/kg bw[per 8 h].

 

Under this condition, the daily dose is about 1/25 of the estimated threshold dose (single, i.p.) in rodents of 200 mg/kg bw. Despite repeated dosing, it can be anticipated that the GSH pool in the gonads will not be affected to significant extent due to capacities of recovery and compensation. On the other hand, this concentration proved to be acutely irritating in the nasal region of rat and is likely to produce chronic nasal inflammation and possibly neuroblastomas in the olfactory epithelium of rats. The local effect is supposed to limit the application of exposure concentrations high enough to produce systemic adverse effects including the reproductive system. Note: The highest concentration applied in the 90d inhalation study (Coombs et al. 1993) was 300 mg/m3(= 60 ppm), which corresponds to about 30 mg/kg bw/d [6 h/d]. No macroscopic and microscopic lesions were noted in the male and female reproductive organs. On the other hand, this dose produced massive nasal inflammation, however, no systemic adverse effects.

The findings clearly show that under experimental inhalation conditions the potency of systemic toxicity of naphthalene is insignificant. Likewise – in synopsis with the metabolism and mode of action of naphthalene - this can be assumed for reproductive performance in rodents.

For exposed humans at workplaces, it can be anticipated that there is neither an unreasonable risk for systemic toxicity including the reproductive system at and below the unofficial OEL of 50 mg/m3, for a long time accepted as guidance value in occupational settings, but under discussion since the classification as carcinogen, suspended and/or down-regulated by EU member states.

 

·        Oral administration

Significantly higher doses can be achieved by oral dosing.

But firstly,it has to be considered that the key health effect observed in humans during naphthalene´s long history (also as consumer product) is haemolytic anaemia, an effect that cannot be reproduced in rodents and other species. The validity of a fertility study may be heavily compromised if dosing reaches high levels that may induce haemolytic anaemia in humans. 

Secondly,oral exposure is irrelevant for humans, since the consumer is no target group, but only workers who may be exposed to naphthalene. The relevant route of exposure to naphthalene, taking into account its non-intermediate uses (only relevant uses regarding exposure of workers) is inhalation. Naphthalene sublimes at room temperature which is the reason for detectable air concentrations at workplaces. RMMs to avoid general dust generation are implemented in all companies using naphthalene, which means that in reality, oral uptake of naphthalene by workers is very unlikely.

For workers, no scenario for oral exposure is required in REACH, hence no DNELs. Furthermore, REACH Regul., Annex IX no. 8.7.3., column 1 stipulates the route most adequate and relevant for humans has to be selected. This is not the oral one.

 

Relevance to humans of the rat model

Metabolism in rodents is chiefly by P450 oxidation, with subsequent glutathione conjugation, as well as epoxide hydroxylation to naphthalene 1,2-dihydrodiol. Glutathione conjugation has not been shown to occur in the liver of non-human primates: In Wistar rats, GSH conjugation was found to be dominant in the liver, while naphthalene was excreted as mercapturic acid into the urine, the GSH level decreasing to 17 % of the control the same time. On the other hand, in chimpanzees, naphthalene was excreted as glucuronate or sulphate rather than as mercapturate, while there was no change of the GSH pool under naphthalene stress (Summer et al. 1979: see entry Toxicokinetics).

 

Naphthalene failed to increase the excretion of urinary mercapturic acids and biliary GSH conjugates of naphthalene in chimpanzees. This is in agreement with findings of Boyland and Sims (1958) who detected only “traces” of mercapturic acids in the urine of man after the application of a single dose of 500 mg naphthalene to humans.

 

The results suggest that the chimpanzee rather than the rat is a relevant model for man to study detoxification of naphthalene, hence to study particular toxic endpoints. Similar doubts have been raised about whether the rat represent a valid model for the development of naphthalene-induced tumors in the nasal-olfactory region in humans (compare CCSG 2012: Mode of action of naphthalene: see entry Toxicokinetics).

 

Worker health study

Downstream users of naphthalene performed a human study evaluating exposed workers in the abrasive industry (industry expected to have highest exposure levels) from July to October 2014. The final report is currently still being discussed and revisited, expected to be finalised before summer vacation 2016. Exposure concentrations were monitored along with the determination of biomonitoring data in urine as well as the histological and biochemical investigations into the exposure-related signs of inflammation of the respiratory tract. Information derived from this study is considered to be relevant for decisions about further preventive/precautionary actions to be taken or not.

 

Independent expert opinions

SCOEL and the authors of the available EU Risk Assessment Report also evaluated the available scientific information as well as the general potential risk for impacts on reproduction caused by workers exposure to naphthalene:

In their 'Recommendation from the Scientific Committee on Occupational Exposure Limits for naphthalene' published in March 2010, SCOEL came to the conclusion: “Overall, there areno grounds to indicatethat naphthalene wouldadversely affect fertility.… it appears that naphthalenedoes not show developmental toxicityat doses which are not maternally toxic … .' (SCOEL 2010, p. 6).

In the EU Risk Assessment Report for naphthalene published in 2003, the rapporteurs concluded that 'There are no concerns for irritation, sensitisation, mutagenicity or for effects on reproduction'.

 

In their evaluation of the EU Risk Assessment Report, the SCTEE (SCIENTIFIC COMMITTEE ON TOXICITY, ECOTOXICITY AND THE ENVIRONMENT) (2002) stated that‘No fertility study with naphthalene has been reported. Changes in the reproductive organs have not been detected in repeated dose studies, but the absence of data on reproductive function indicates the need for a two-generation reproductive study’

In their reply to SCTEE, HSE (Ball 2002) expressed their‘concern over the validity of the animal model for conducting a specific fertility study given that the lead health effect identified in humans is haemolytic anaemia - a toxicological endpoint not adequately demonstrated in animal models. Hence, on this basis, the relevance of the findings of a dedicated fertility study with respect to making predictions on potential reproductive toxicity to humans could be open to question.’

 

Ongoing regulatory actions

Naphthalene has been selected for CoRAP inclusion by Germany based on the following arguments, which will be subject of the substance evaluation in 2016: ‘An ongoing scientific debate has identified inflammatory reactions in the olfactory epithelium as the most critical effect.' Toxicity to reproduction has not been identified as a concern for naphthalene by the CA. Furthermore, naphthalene is classified as carcinogenic. Consequently, in case workers are sufficiently protected from these effects, it can be expected that also workers are sufficiently protected by implemented RMMs from potential reproductive effects.

 

Conclusion

Against this background, the registrant believes that there is sufficient information (weight of evidence) in the toxicity database for naphthalene to assume that naphthalene does not cause reproductive effects. In summary, the results of all available repeated-dose toxicity studies, developmental toxicity studies, toxicokinetic information along with the concrete doubts about the validity of the rodent model being relevant to humans allow the conclusion that any generation-reproduction study is not justifiable based on scientific weight of evidence.

 

For the time being, the scientific debate concentrates on inflammatory reactions in the olfactory epithelium as the most critical effect. Since recently, this aspect has been scrutinised in detail by Rhomberg et al. 2010 and Bailey et al. 2016 (see also Toxicokinietics and Mode of Action).

 

Reference:

SCOEL 2010:Recommendations from the Scientific Committee on Occupational Exposure Limits for Naphthalene. Scientific Committee on Occupational Exposure Limits, European Commission, SCOEL/SUM/90, March 2010

ECB 2003:European Union Risk Assessment Report NAPHTHALENE [CAS No: 91-20-3; EINECS No: 202-049-5] RISK ASSESSMENT European Communities, 2003 [http://ecb.jrc.ec.europa.eu/esis/]

SCTEE 2002:Opinion on the results of the Risk Assessment of NAPHTHALENE. Scientific Committee on Toxicology, Ecotoxicology and the Environment (CSTEE) [SCTEE-out137_en]

Ball 2002:Reply to CSTEE comments on the results of the Risk Assessment of Naphthalene. HSE/UK

Bailey, L.A.; Nascarella, M.A.; Kerper, L.E.; Rhomberg, L.R. (2016):Hypothesis-based weight-of-evidence evaluation and risk assessment for naphthalene carcinogenesis. Crit. Rev. Toxicol. 46: 1-42

Rhomberg, L.R.; Bailey, L.A.; Goodman, J.E. (2010):Hypothesis-based weight of evidence: A tool for evaluating and communicating uncertainties and inconsistencies in the large body of evidence in proposing a carcinogenic mode of action—naphthalene as an example. Crit. Rev. Toxicol. 40: 671-696


Short description of key information:
No generation reproduction studies investigating effects on fertility are available. A Weight-of-Evidence approach is undertaken to fill this data gap.

Effects on developmental toxicity

Description of key information
Naphthalene orally administered to rats and rabbits induced no developmental toxicity at up to maternally toxic doses. At the highest dose in rats (450 mg/kg bw/d), distinctly toxic to the dams, a trend to some foetotoxicity became apparent.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 27. 1990 to February 20. 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP conform study following modern procedures.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Inc., Raleigh, NC
- Age at study initiation: 8-10 weeks
- Weight at study initiation: 209-271 g
- Housing: individually housed in solid-bottom polycarbonate cages with stainless steel wire lids
- Diet: Ground Purina Certified Rodent Chow (#5002) ad libitum
- Water: Deionized/filtered water ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 72°F (range 69-75°F) [22°C (range 20.5-24°C)]
- Humidity (%): 55% (range 49-60%) and 48% (range 44-63%).
- Photoperiod (hrs dark / hrs light): lights on from 06:00h to 18:00h
Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
Mazola
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For both study replicates, each dose of NAP was formulated independently in a sufficient quantity to last the entire dosing period. The chemical/vehicle mixtures had to be refrigerated to insure stability. To avoid repeated heating and cooling of the NAP solutions, each dose formulation was divided into aliquots, such that only one aliquot of each formulation was needed for any particular day of dosing. Pre- and post-dosing analysis performed by RTI of the NAP dose formulations for each replicate were within 96%-108% of their theoretical concentrations. Thus, the NAP/vehicle formulations were considered to be stable throughout the period of use for each study replicate.

VEHICLE
- Concentration in vehicle: The actual dose delivered to each animal was adjusted daily, according to a weight taken prior to dosing.
- Amount of vehicle (if gavage): 5 ml/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Pre- and post-dosing analysis performed by RTI of the NAP dose formulations for each replicate were within 96%-108% of their theoretical concentrations.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: 1 night
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: Sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
On gestational days 6 through 15
Frequency of treatment:
Daily
Duration of test:
20 days
Remarks:
Doses / Concentrations:
0, 50, 150 and 450 mg/kg body weight/day
Basis:
analytical conc.
No. of animals per sex per dose:
28
Control animals:
yes, concurrent vehicle
yes, historical
Details on study design:
- Dose selection rationale:

The dose range selected for this study was based on preliminary data furnished by EHRT (NTP, 1990). EHRT studied the effects of naphthalene (0, 100, 400, 500, 600 or 800 mg/kg/day administered by gavage on gestational days 6 through 15) on a limited number of maternal and fetal endpoints. Significant maternal and fetal toxicity was observed at 600 and 800 mg/kg/day. For example, 67% of the dams died during treatment in the 800 mg/kg/day dose group and of those dams that survived the treatment, 33% were found to have totally resorbed their litters. Further, slight maternal and fetal toxicity was observed for dams in the 400 and 500 mg/kg/day groups, indicating that the maternal LD50 for naphthalene was between 400 and 500 mg/kg/day. Since it was desirable for the highest dose used in the current study to cause less than 10% maternal death and because the maternal LD10 for naphthalene could not be determined from the EHRT study, 450 mg/kg, a dose midway between the 400-500 mg/kg doses was chosen. The lower doses were selected so that a no effect level could be established for both maternal and fetal toxicity.
Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 3, 6-15, 18, 20

FOOD CONSUMPTION: Yes
- 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

WATER CONSUMPTION: Yes
- Time schedule for examinations: On days 0, 3, 6, 9, 12, 15, 18, and 20.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20.
- Organs examined: The maternal body, liver, and intact uterus were weighed and corpora lutea were counted. Uterine contents were examined to determine the number of implantation sites, resorptions, dead fetuses and live fetuses. Uteri which had no visible implantation sites were stained with ammonium sulphide (10%) to detect very early resorptions.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter
Statistics:
General Linear Models (GLM) procedures, were applied for the analyses of variance (ANOVA) of maternal and fetal parameters. Prior to GLM analysis, an arcsine-square root transformation was performed on all litter-derived percentage data and Bartlett's test for homogeneity of variance was performed on all data to be analyzed by ANOVA. GLM analysis determined the significance of dose-response relationships and the significance of dose effects, replicate effects and dose x replicate interactions. When ANOVA revealed a significant (p<0.05) dose effect, Williams' and Dunnett's Multiple Comparison Test compared NAP-exposed to control groups. One-tailed tests were used for all pairwise comparisons except maternal body and organ weights, maternal food and water consumption, fetal body weight and percent males per litter. Nonsignificant (p>0.05) dose x replicate effects on selected fetal parametric measures were considered justification for pooling data across replicates for nonparametric analysis on related measures. When a significant (p<0.05) dose x replicate interaction occurred, the data for that endpoint and for any related nominal scale data were analyzed separately for dose effects within each replicate in the study, as well as for all replicates combined. Nominal scale measures were analyzed by a X2 test for independence and by a test for linear trend on proportions. When a x2 test showed significant group differences, a one-tailed Fisher's exact probability test was used for pairwise comparisons of NAP and control groups.
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: Clinical signs of toxicity, reduced food/water consumption, reduced body weight

Details on maternal toxic effects:
Three dams had to be removed from the study. One dam in the 150 mg/kg/day-group was removed due to urinary calculi, a condition which probably existed prior to the initiation of dosing. In the 450 mg/kg/day group, one dam was removed because it delivered on day 20 and another dam in that group was removed because it had microphthalmia, a condition which existed prior to the start of dosing. Two dams died during dosing and both were in the 50 mg/kg/day group. Necropsy data indicated that the deaths were unrelated to dosing errors.

NAP-treated animals exhibited clinical signs of toxicity which were observed in all treatment groups on the first day of dosing. Lethargy, slow breathing, and prone body posture were the most common clinical signs during the first five days of dosing, with the incidence of these effects showing a marked dose-dependence. For example, on gd 6, the first day of dosing, 81% of the dams in the 50 mg/kg/day group exhibited one or more of the above clinical signs as compared to 96% for both, the 150 and 450 mg/kg/day groups. By the third day of dosing, the clinical signs of toxicity had essentially disappeared in the 50 mg/kg/day group, indicating that the dams had acquired tolerance to these effects of NAP. A similar trend was noted for 150 mg/kg/day NAP, but tolerance was not noted until the sixth day of dosing (gd 11). In the 450 mg/kg/day group the incidence of NAP-induced lethargy and slow respiration also declined during the dosing period, but it never fell below 15%. As the rats became tolerant to the depressant effects of NAP, the incidence of rooting behavior increased; rooting behavior is commonly observed in rodents following gavage administration of compounds which have a noxious odor or which act as a local irritant. The occurrence of rooting behavior, which was observed in less than 10% of the dams during the first few days of dosing, gradually increased as dosing progressed. By gd 15, 24% of the dams in the 150 mg/kg/day group and 92% of the dams in the 450 mg/kg/day group exhibited rooting behavior, as compared to 0% for both, the control and 50 mg/kg/day NAP groups.

NAP also reduced maternal food and water consumption in the two highest dose groups. On gd 6 to 9, relative food intake was suppressed 25% by 150 mg/kg NAP and 37% by the higher dose. However, by gd 9 to 12, food consumption in the two groups had returned to control levels. A significant elevation in food consumption was noted on gd 18 to 20 in the 450 mg/kg/day group. Relative water intake in both groups displayed a pattern similar to food consumption. From gd 6 to 9, a 19% decrease was observed in the 150 mg/kg/day group (significant) and a 13% reduction in the 450 mg/kg/day group (nonsignificant); water consumption in both groups showed significant elevations of 15%-25% on gd 9 to 12 through gd 15 to 18, be-fore returning to control levels by gd 18 to 20. Absolute food and water consumption were similarly affected.

NAP also caused a significant dose-dependent decrease in maternal body weight and body weight gain; the effect was confined to the 150 mg/kg and 450 mg/kg groups, the same groups which exhibited suppressed food and water consumption. In these two groups, dams actually lost weight during the first three days of dosing, coincident with the time when nutrient intake displayed its greatest decreases in those animals. After gd 9, when food and water consumption were at or above control levels, the rats in the 150 mg/kg/day and 450 mg/kg/day groups gained weight at nearly the same rate as controls. Nevertheless, weight gain from gd 6 to 15 was decreased 31% and 53% relative to controls in the 150 and 450 mg/kg/day groups respectively. This indicated that the rats were not able to offset the initial NAP-induced weight deficits. Weight gain from gd 0 to 20 was also significantly less than controls (13-20%) in these two groups. Thus, even after the dosing period had ended, the dams were not able to fully recover from the earlier effects of NAP. As a result of the effects on weight gain, maternal body weights in the two groups remained consistently below control values (6-12%) throughout dosing and this trend persisted until sacrifice on gd 20. The adverse effects of 150 mg/kg/day and 450 mg/kg/day NAP on maternal weight and weight gain were not secondary to decreased uterine weight because deficits were observed for both corrected and absolute weight gain from gd 0 to 20.

Maternal liver weights (absolute or relative) were comparable across groups. There was a trend toward decreased gravid uterine weight with increasing dose of NAP. Gravid uterine weights of the 50, 150, and 450 mg/kg/day groups were 105%, 95% and 89% of control values respectively, but
there was no significant difference among groups by ANOVA.
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
LOAEL
Remarks:
(highest dose tested)
Effect level:
450 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
LOAEL
Remarks:
(lowest dose tested)
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Results from the uterine examination revealed that the number of corpora lutea per dam and the number of implantation sites per litter in the NAP-treated dams were within 95-102% of control values and the number of live fetuses per litter was likewise unaffected. There were also no statistically significant effects of treatment upon the incidence of resorptions or late fetal deaths. However, there was a significant trend test for the percent adversely affected implants per litter. The incidence of conceptuses adversely affected (ie. non-live or malformed) was 8%, 7%, 13% and 20% in the control through high-dose groups, respectively. Average fetal body weight per litter also exhibited a significant trend test. Fetal body weights in the 50, 150, and 450 mg/kg/day groups were 105%, 99% and 95% of control values respectively. However, ANOVA did not detect a significant overall effect of dose for either the percent adversely affected fetuses or the average fetal body weight per litter.

Analysis of anatomical defects indicated that NAP treatment tended to increase the incidence of malformations. Both the percent fetuses malformed per litter (4%, 4%, 7% and 10%) and the percent litters with malformed fetuses (23%, 27%, 33% and 50%) showed a significant trend test. The largest difference was seen in the 450 mg/kg/day group where the percent fetuses malformed per litter were 2.5 times greater than controls (ie. 10% vs.4%). Nevertheless, ANOVA did not detect a significant effect of dose for the percent malformed fetuses per litter, nor was there a significant difference among groups for the percent litters with malformations by the Chi-Square Test.

A review of the individual malformations suggested an effect of treatment on the incidence of enlarged ventricles of the brain. Indeed, separate statistical analysis of this malformation indicated that there was a significant dose-dependent increase in the percent fetuses per litter and the percent litters with enlarged ventricles (significant trend test for both). For example, the percent fetuses with enlarged ventricles in the 50, 150, and 450 mg/kg/day groups was 1.1-, 1.6-, and 2.8-fold higher than control values respectively. However, the main affect for dose in the ANOVA was not significant and a significant effect of replicate was detected for this measure. In the first replicate, there was no effect of treatment on the incidence of enlarged lateral ventricles, with 9% of the fetuses per litter affected in the 450 mg/kg/day group versus 6% in the controls. In contrast, the percentage of fetuses per litter in the second replicate displaying this malformation was only 0.6% in the controls, as compared to 9% in the 450 mg/kg/day group (significant). Thus, the overall effect of treatment on this particular malformation (and malformations in general) is confounded by a 10-fold change in the incidence of this malformation between replicates within the control group.

In contrast to the effect of NAP on malformations, the overall incidence of variations showed a decreasing trend. The percent fetuses with any variation per litter was 45% lower than controls in both the 150 and 450 mg/kg/day groups, but the main effect for dose in the ANOVA was
nonsignificant.
Abnormalities:
not specified
Developmental effects observed:
not specified

Table 1. Maternal Toxicity in Sprague-Dawley Rats exposed to Naphthalene on Gestational Days 6 through 15

 Naphthalene (mg/kg/day)

0

50

150

450

Subjects (dams)

 

 

 

 

    Total Treated

28

28

28

28

    No. Removed or dead

0

2

1

2

    No. (%) pregnant at sacrifice

26 (93%)

26 (93%)

25 (93%)

26 (100%)

Maternal Body Weight Gain (g)(±SEM)

 

 

 

 

    Treatment period (gd 6 – 15)

55.1 ± 2.0§

53.2 ± 3.1

37.8 ± 2.4*

26.0 ± 3.1*

    Gestation period (gd 0 – 20)

161.0 ± 3.7§

161.3 ± 4.2

140.5 ± 4.8*

129.3 ± 5.6*

    Corrected gestation bw gain

(-gravid uterus)

              

77.0 ± 3.4§

73.1 ± 3.3

60.4 ± 3.0*

54.5 ± 2.8*

    Gravid uterine weight

84.0 ± 2.4§

88.2 ± 3.8

80.2 ± 3.7

74.8 ± 5.2*

Maternal liver weight

 

 

 

 

    Absolute (g)

17.4 ± 0.3

16.8 ± 0.3

16.4 ± 0.4

16.6 ± 0.4

    Relative (% body weight)

4.36 ± 0.05

4.23 ± 0.06

4.34 ± 0.07

4.54 ± 0.09

* Dunnett’s or William’ test, p<0.05

§Test for linear Trend, p<0.05

 


Table 2. Developmental Toxicity in Sprague-Dawley Rats Following Maternal Exposure

to Naphthalene on Gestational Days 6 through 15

Naphthalene (mg/kg/day)

All data on per litter basis (±SEM)

0

50

150

450

All litters (No.)

26

26

25

26

    No. corpora lutea per pregnant dam

15.2 ± 0.4

15.5 ± 0.6

14.5 ± 0.7

15.2 ± 0.5

    No. Implantation sites

14.9 ± 0.4

15.1 ± 0.7

14.4 ± 0.6

14.3 ± 0.7

    % Resorptions

3.9 ± 0.9

2.6 ± 0.9

5.8 ± 4.0

11.3 ± 5.5

    % Litters with one or more resorption

50

31

32

27

    % Late fetal deaths

0.2 ± 0.2

0.0 ± 0

0.3 ± 0.3

0.0 ± 0

    % Litters with one or late fetal death

4

0

4

0

    % Nonlive implants per litter

4.2 ± 0.9

26 ± 0.9

6.1 ± 4.0

11.3 ± 5.5

% Litters with one or more nonlive implants

54

31

36

27

    % Litters with 100% nonlive implants

0

0

4

8

    % Adversely affected implants

8.2 ± 1.9 §

6.8 ± 2.3

13.1 ± 4.4

20.2 ± 5.6

% litters with one or more adversely affected implants

65

50

56

65

Live Litters

26

26

24

24

    No. Live fetuses

14.3 ± 0.5

14.7 ± 0.7

14.5 ± 0.4

14.3 ± 0.7

    Average fetal body wt (g)

 

 

 

 

         Male fetuses

3.8 ± 0.05 §

4.00 ± 0.12

3.74 ± 0.06

3.61 ± 0.06

         Female fetuses

3.62 ± 0.04 §

3.71 ± 0.06

3.63 ± 0.07

3.45 ± 0.06

    % Male fetuses

56 ± 3

55 ± 3

51 ± 2

50 ± 3

Malformations

 

 

 

 

    % Fetuses malformed

4.1 ± 1.9 §

4.3 ± 2.2

7.5 ± 2.6

10.0 ± 3.0

         Male fetuses

4.7 ± 2.1

3.7 ± 1.5

6.7 ± 3.0

10.4 ± 3.6

         Female fetuses

3.8 ± 2.1 §

4.1 ± 3.1

7.4 ± 2.9

9.7 ± 3.0

    % Litters with malformed fetuses

23

27

33

50

    % Fetuses with enlarged ventricles (brain)

 

 

 

 

         Replicate I

5.9 ± 3.4

7.1 ± 4.2

7.6 ± 3.7

8.8 ± 4.3

         Replicate II

0.6 ± 0.6 §

0.0 ± 0

2.1 ± 2.1

8.9 ± 4.4 *

Variations

 

 

 

 

    % Fetuses with variations

18.9 ± 4.2 §

19.9 ± 4.0

10.4 ± 3.5

10.3 ± 2.8

    % litters with one or more variations

81

69

42 **

58

* Dunnett’s or William’ test, p<0.05

** Fisher’s Exact Test,p<0.05

§Test for linear Trend, p<0.05

Conclusions:
In conclusion, naphthalene was not fetotoxic or teratogenic. There is some evidence that minimal developmental toxicity with an increasing trend of visceral malformation occurs at clearly maternally toxic doses.

Executive summary:

In this well conducted study, groups of 28 female Sprague-Dawley rats were treated by gavage with 0, 50, 150 or 450 mg/kg/day naphthalene on days 6-15 of gestation. Caesarean sections were performed on day 20. The results from this study indicate that naphthalene administered orally had minimal effects on fetal development in the presence of significant maternal toxicity.

Developmental toxicity was expressed only as significant trends toward decreased fetal weight and increased incidence of visceral malformations in the naphthalene-treated groups. The dose of naphthalene which caused the largest developmental changes (450 mg/kg/day) also significantly reduced maternal corrected weight gain and food consumption.

There was an increase in the incidence of one visceral malformation (enlarged lateral ventricles of the brain), but only in one replicate in the 450 mg/kg/day group. It is equivocal whether this represents a direct effect of naphthalene on fetal development, because there was an order of magnitude difference in the incidence of these malformations between replicates in the control group. Additionally, the incidence of enlarged lateral ventricles of the brain in the background control data from RTI laboratory has been reported to be highly variable in recent years. In six NTP-sponsored rat developmental teratology studies conducted at RTI from January 1989 through December, 1990, the occurrence of this particular malformation has ranged from 0% to 27%.

Therefore, apparent developmental toxicity of naphthalene cannot be based solely on an increase in the incidence of enlarged lateral ventricles in one replicate of the high dose group. Therefore, it can be concluded a developmental NOAEL for orally administered naphthalene is closer to 450 mg/kg/day and the developmental LOAEL is probably higher than 450 mg/kg/day naphthalene. In the absence of other data, 150 mg/kg bw/d has been adopted as unequivocal NOAEL.

Naphthalene caused significant maternal toxicity. Two dams died during dosing in the 50 mg/kg/day group, but the absence of any deaths in the higher dose groups does not suggest an association between naphthalene-treatment and maternal mortality.

More consistent were the significant reductions in maternal body weight and weight gain in the 150 mg/kg/day and 450 mg/kg/day naphthalene groups. The effects of these two doses on maternal weight parameters may be secondary to naphthalene-induced suppression of food and water consumption on gd 6 to 9. The fact that maternal body weight began to rise in these two groups at essentially the same rate as controls after food and water consumption returned to normal, supports this view. Furthermore, when maternal nutritional status was not affected by treatment (50 mg/kg/day naphthalene), no effect on maternal weight or maternal weight gain was observed. The threshold for effects of naphthalene on respiration and motor activity was lower than that for weight reduction since 50 mg/kg/day naphthalene was comparable to 450 mg/kg/day naphthalene in producing shallow breathing and lethargy during the early phases of dosing.

The LOAEL for naphthalene-related maternal toxicity is 50 mg/kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
high
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

With respect to developmental toxicity, the only information available in humans comes from cases of haemolytic anaemia in infants born to mothers also suffering haemolytic anaemia, following ingestion of unquantified doses of naphthalene during their pregnancy demonstrating that naphthalene and/or its metabolites can cross the placental barrier.

Inhalation or dermal developmental toxicity studies in animals are not available.

Acute oral exposure of pregnant rats to naphthalene doses of 150 or 450 mg/kg/day (but not 50 mg/kg/day) during gestation has produced maternal toxicity including clinical signs (lethargy and prone position) and severe decreases in body weight gain, but clear effects on the developing foetus were not found at maternal oral doses as high as 450 mg/kg/day in rats or 120 or 400 mg/kg/day in rabbits.

In summary, in rats, some foetotoxicity but no malformation was observed at doses causing significant maternal toxicity (450 mg/kg/day). In rabbits, no developmental effects were seen in one study at a dose causing mild maternal toxicity, or in another study at a dose close to those producing pronounced maternal toxicity. Overall, naphthalene only produces foetotoxicity at maternally toxic doses in animals, and does not produce developmental toxicity at maternally subtoxic doses.


Justification for selection of Effect on developmental toxicity: via oral route:
Key study with lowest NOAEL (foetotoxicity), while study in rabbit without adverse effects

Justification for classification or non-classification

As results of animal studies demonstrate that naphthalene only produces foetotoxicity at maternally toxic doses in animals, and does not produce developmental toxicity at maternally subtoxic doses, it can be concluded that there are no classification obligations.

Additional information

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