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Endpoint summary

Description of key information

This category of UVCB substances (streams) are generally made by similar manufacturing processes involving steam cracking. Consequently, they have many of the same constituents within the stream compositions. The proportion of these constituents will vary. The major groups of constituents within the resin oils and cyclic dienes are unsaturated non-hydrotreated products (the resin oil products) dicyclopentadiene (DCPD) and methylcyclopentadiene-cyclopentadiene; as well as indane and indene. These streams may also have variable concentrations of aromatics, including benzene, styrene, toluene, xylenes and naphthalene. These notable mono-constituents are referred to as the “marker substances”. See the Category Justification Document (CJD) attached in Section 13.2 for full detail.

To support the category approach adopted by LOA for these UVCB streams, coherence and read-across between the UVCBs in the category, the following approaches were taken:

1. Full category coverage of UVCB streams and marker substances. UVCB streams have been coded L-01 to L-12, and the marker substances that were considered toxicologically relevant were benzene, xylene, naphthalene, DCPD, cyclohexane, indene, ethylbenzene, toluene and styrene. All of these UVCB streams and markers (except ethylbenzene, toluene and styrene) were evaluated in a 14-day repeated-dose toxicity study, according to the parameters of the OECD 407 repeated-dose oral toxicity study in rodents adopted October 2008. Additionally, terminal plasma was collected for metabolomic analysis. Metabolomics is a new approach methodology explored by LOA to determine whether the technology is useful to support complex UVCB category grouping and read-across. The doses for these studies were selected from 7-day dose-range finding studies conducted prior to the main studies.
2. OECD 422 combined repeated-dose toxicity and reproduction/developmental toxicity screening studies on selected representative streams aimed at covering the compositional breadth of the category. To ensure efficient use of animals, the doses for these studies were selected based upon the results from the 14-day metabolomics studies.
3. These data are supported by available 90-day repeated-dose toxicity studies (OECD 408) on marker substances.

All of these data are summarised and discussed below in the “Additional Information” section

Additional information

There are currently 21 x 14-day repeated-dose toxicity studies:

 

• 12 x Category L UVCB substances


• 3 x BASF UVCB substances that are consistent with L-01, L-02 and L-03 (these are used as “controls” for the UVCBs)


• 6 x Marker substances (indene, cyclopentane, dicyclopentadiene, naphthalene, xylene and benzene). Additionally, ethylbenzene, toluene and styrene data have been obtained from pre-existing 28-day repeated-dose toxicity studies in which classical toxicology parameters and metabolomics data have been collected. A justification for why it is appropriate to include these data is included in Section 13.2

 

There are 6 x OECD 422 combined repeated dose toxicity study with the reproduction/developmental toxicity screening studies which characterise the compositional space within the Resin Oils and Cyclic Dienes category (Category L – further information on this is available in the testing strategy document attached in Section 13.2).

 

In addition, there are 90-day repeated-dose toxicity study for the marker substances DCPD, benzene and toluene, and an OECD 422 combined repeated dose toxicity study with the reproduction/ developmental toxicity screening study for DCPD.

 

 

Data Discussion - All Studies

 

 

Doses

 

	Doses			NOAELs
	14-day (mg/kg/day)	OECD 422 (mg/kg/day)	90-day (mg/kg/day)	14-day NOAEL M&F (mg/kg/day)	422 NOAEL M&F (mg/kg/day)	90-day NOAEL M&F (mg/kg/day)
Indene	100, 450	-	-	100	-	-
Cyclopentane	300, 1000	-	-	1000	-	-
DCPD	50, 150	4, 20, 100	10, 30, 100	50 M, <50 F	4 M, 20 F	30
Napthalene	250, 600	-	-	<250 M, 250 F	-	-
Xylene	300, 1000	-	-	300	-	-
Benzene	300, 1000	-	25, 50, 100, 200, 400, 600	<300	-	100 M, not established F
Toluene	-	-	312, 625, 1250, 2500, 5000	-	-	625
L-01	300, 1000	-	-	<300 M, 300 F	-	-
L-02	250, 750	60, 200, 600	-	<250 M, 250 F	600	-
L-03	150, 500	30, 100, 300	-	<150	300	-
L-04	70, 200	25, 70, 200/140	-	70	70	-
L-05	250, 750	60, 200, 600	-	250	600	-
L-06	300, 1000	-	-	300 M, <300 F	-	-
L-07	200, 600	-	-	<200 M, 200 F	-	-
L-08	100, 300	-	-	100	-	-
L-09	70, 200	5, 25, 100	-	70	5	-
L-10	300, 1000	-	-	300	-	-
L-11	150, 500	35, 125, 375	-	150 M, <150 F	35	-
L-12	300, 1000	-	-	300 M, <300 F	-	-

 

Liver

 

Only a few target organs could be identified by classical parameters from clinical pathology as well as histopathology. Parameters for liver cell dysfunction were observed in clinical pathology for indene, DCPD, naphthalene, and xylene as well as in most LOA streams. Furthermore, increased alanine transaminase (ALT) levels, indicative of liver cell membrane degradation, were observed for naphthalene, L-01, L-02, L-03, L-05, L-06, and L-07. Increased liver weights were generally observed in both sexes for all compounds at the high dose treatment, except for benzene or cyclopentane. For some of the test streams, liver weight increases were also seen at the low dose for L-01, L-02, L-04, L-05, L-06, L-07, L-11, L-12, and naphthalene. The histopathological examinations revealed hepatocellular hypertrophy (centrilobular, periportal, and/or diffuse) as the main histological finding. This finding was not observed for the marker compounds with the exception of xylene in male animals, or L-09, benzene, cyclopentane, and indene in female animals. Benzene showed single cell necrosis in hepatocytes of female animals at the high dose as well as the low dose. Furthermore, single cell necrosis was observed in individual animals in the high dose groups of dicyclopentadiene (females), naphthalene (females), and L-09 (males).

 

In the OECD 422 studies, UVCBs that represent streams L-02, L-03, L-04, L-05, L-09 and L-11, consistent with observations from the 14-day repeated-dose toxicity studies, liver weight increases and hepatocellular hypertrophy are seen in all UVCBs tested. However, accompanying evidence of markers of liver cell membrane degradation were only seen in L-02 and L-03. Effects observed are generally consistent with the 90-day repeated-dose toxicity studies conducted for the marker substances except for benzene, for which no liver weight increase is reported.

 

Examining changes to the rat metabolome there were metabolic signatures consistent with liver enzyme induction and effects of toxicity for all marker substances tested except for cyclopentane, indene, and naphthalene. All of the LOA UVCB streams and the BASF LOA streams, except L-05 and L-12, showed metabolome signatures consistent with effects observed in the liver. Importantly, there were no metabolome effects observed in the UVCB streams that were not also observed in marker substances. Overall, these data are relatively consistent and support the category and read-across proposed. Full details are available in the RSS for each substance in this dossier, and the metabolomic RSS is attached in section 7.9.4.

 

Thyroid

 

The metabolome data also revealed patterns for indirect thyroid effects (increased excretion of thyroid hormones due to the liver enzyme induction) showing (weak) matches. The pattern for indirect thyroid effects (increased excretion of thyroid hormones due to the liver enzyme induction) is clearly distinguishable from patterns caused by direct thyroid toxicants such as methimazole or propylthiouracil. These direct thyroid toxicants do not match with the patterns for indirect thyroid effects (increased excretion of thyroid hormones due to the liver enzyme induction). The (weak) matches with patterns indicative for liver and/or indirect thyroid effects were detected for benzene (HD), cyclopentane (LD), dicyclopentadiene (HD), indene (HD and LD), naphthalene (HD and LD), xylene (HD), toluene (HD and LD), styrene (HD), ethylbenzene (HD), BASF LOA 1 (HD and LD), BASF LOA 2 (HD and LD), BASF LOA 3 (HD and LD), L-01 (HD), L-02 (HD), L-03 (HD and LD), L-04 (HD), L-05 (HD), L-06 (HD and LD), L-07 (HD and LD), L-08 (HD), L-09 (HD), and L-11 (HD and LD). No (weak) matches with these patterns were observed for L-10 and L-12.

 

Examining thyroid parameters in the OECD 422 studies, L-02, L-04, L-05, L-09, L-11 and L-03, thyroid organ weights were increased in L-02 and L-05; follicular cell hypertrophy was observed in L-02, L-04, L-09 and L-11, however accompanying changes in thyroid hormones T3, T4 and TSH were generally minor and inconsistent across the OECD 422 studies. While the thyroid hormone data from the OECD 422 do not exactly mirror those observed in the 14-day repeated-dose toxicity (metabolomics studies), there is histological evidence in the OECD 422 studies describing thyroid hypertrophy in L2, L4, L9 and L11 (not L-03) and while not observed in L-05, there was increased thyroid weight in L-05. Increased thyroid weight is commonly attributed to hypertrophy and/or hyperplasia of the thyroid, and would be commonly expected in the presence of thyroid weight increase. Overall, the data are consistent between the 14-day repeated-dose toxicity metabolomics studies and the OECD 422 study results that streams from the resin oils and cyclic dienes category affect thyroid gland homeostasis via a mechanism secondary to the increased liver weights and associated enzyme induction. There is no evidence in either study type for direct effects on thyroid homeostasis.

 

Kidney

 

There is consistent data in males in the both the OECD 422 study and the 14-day metabolomic study with kidney toxicity presenting as increased severity of hyaline droplet accumulation, tubular basophilia and granular casts. Immunohistochemistry evaluation of the kidneys revealed α2u-globulin accumulation in male rat kidneys in all instances. This mechanism is known to be rat specific and is of no consequence to humans in this instance.

 

Clinical pathology and haematology

 

In clinical pathology, regenerative anaemia and effects on the red blood cell metabolism were observed for indene, naphthalene and most of the LOA streams (with the exception L-04, and L-09). Furthermore, indene, naphthalene and L-02, L-03, L-05, L-07, and L-09 showed signs of an acute phase reaction. In contrast benzene induced a lymphocytopenia[CH1] . Some LOA streams showed an imbalance of electrolyte/minerals: L-01, L-02, L-03, L-04, L-05, L-08, L-09, and L-12. No adverse effects were observed for cyclopentane in both sexes, L-04 in male animals, and L-10 in female animals. Occasional weight changes were observed in other organs, such as spleen, heart, adrenals, ovaries, testes, seminal vesicle (except: L-11, L-12; a treatment-related effect cannot be excluded), epididymides, and prostate (except: L-09, L-11, L-12; a treatment-related effect cannot be excluded) were at least partially related to body weight changes and not considered treatment-related. For L-03, L-04, and L-07, alveolar histiocytosis was observed in some male animals. Since this finding is also occasionally seen in control animals and the finding was only minimal it was regarded to be not adverse. Further, treatment-related histological changes were not observed for lung, nor for ovaries or testes in these LOA streams. Furthermore, the prostate, seminal vesicle and coagulation glands evaluated for L-12 due to reduced organ weights showed no histological changes.

 

In the OECD 422, minor haematological changes were observed in streams L-02, L-03, L-04 and L-05, with the most notable perturbations occurring in stream L-02. In L-02, increases in monocytes, eosinophils, neutrophils, the large unstained count, as well as decreases in the platelet count and haemoglobin were all reported, alongside a decrease in platelet count. This contrasts with the 14-day repeated-dose studies, in which the only haematological changes observed were an increase in reticulocytes, which was not observed at all in the OECD 422 study for L-02 . Red blood cell count was decreased in L-04 and L-03; monocytes were increased in L-03 and L-09; reticulocytes were increased in L-03, L-05 and L-11; HCT was decreased in L-03 and L-04; RDWG was increased in L-04 and haemoglobin as decreased in L-03, L-04, L-09. Extramedullary haematopoesis of the spleen was noted concurrently in L-02, L-03 and L-04 and whilst it was not reported in L-04, enlargement of the spleen itself was. It was concluded that these haematological changes were likely to have been secondary to the observed splenic effects.

 

In the metabolomic study, none of the LOA Category L UVCB streams, nor the marker substances, triggered a response for haematopoesis in the spleen. Despite this being observed in the OECD 422 studies, the incidence was not considered to be adverse. In the 14-day repeated-dose toxicity studies, decreases in haemoglobin were consistent amongst all streams that contained significant concentrations of either indene or naphthalene. These included: L-03, L-06, L-11 and L-12, as well as the marker constituents indene and naphthalene themselves. The L-06 stream was later removed from Category L due to its high naphthalene content, whilst L-12 was removed from the category as the sole registrant ceased production. L-03 and L-11 remain in the category, and are grouped together based upon their compositions containing high concentrations of indene and indane. In the OECD 422 study for L-03, a decrease in haemoglobin, with corresponding decreases in red blood cell count and the haematocrit were observed. Similar effects were not observed in the OECD 422 for L-11, however, the relative concentrations of indene in L-03 and L-11 for the OECD 422 study test materials were 59.9% and 13.8% respectively, whereas in the 14-day repeated-dose toxicity study, where these effects were observed for both, the concentration of indene in L-03 and L-11 was 56.3% and 66.2% respectively.

 

Regenerative anaemia was reported in the 14-day repeated-dose toxicity studies for males in stream L-11, and females in both L-03 and L-11 streams.

 

No other blood-related pathophysiologies were considered relevant, as they did not elicit a positive response in the metabolome following exposure to either the category UVCBs or the relevant marker constituent.

 

Metabolomic data summary and overall conclusion

 

NOTE: All references to figures in this summary of the metabolomic data refer to those included in the metabolomic RSS in Section 7.9.4 (Specific Investigations: LOA metabolomics project).

 

The comparison of the metabolome of the (BASF, LOA 1-2) and LOA-streams as well as the marker compounds showed matches or weak matches with a limited set of patterns. No additional matches with metabolomics patterns were seen for the BASF LOA streams and the LOA-streams that have not also been observed with the tested marker substances. These metabolome patterns were related to effects in liver (with liver enzyme induction as the predominant effect), thyroid (secondary to the liver enzyme induction), and kidney. It has to be noted that no matches were observed with patterns for direct thyroid toxicity. Further (weak) matches were also observed for effects on the blood cell homeostasis for some LOA streams in female animals and for BASF LOA 2 in male animals.

 

Considering the overall strong metabolomic response, it is remarkable, that only a very limited number of patterns were identified as (weak) matches, and that these were predominantly associated with effects on the liver. This is a first indication that from a high-level perspective, the compounds tested were rather similar in their toxicity.

 

The matches for liver, thyroid and kidney effects as well as red blood cell system are in line with the findings in clinical chemistry, organ weight changes and histopathology. It can be concluded that all evaluations show a limited, similar spectrum of toxic effects. Quantitative differences are reflected by the different dose levels used to induce the above-mentioned effects.

 

Statistical analysis

 

Different statistical clustering approaches based on plasma metabolome data were used to identify potential groups of compounds with higher similarity within the streams.

 

The similarity of the test substances based on the profile comparison (treatment correlation analysis) is fairly high. Sub-clusters of higher similarity could be observed for indene, naphthalene, L-06, and L-11, for dicyclopentadiene, L-04, L-08, and L-09, for toluene, ethylbenzene and styrene, for the BASF LOA streams and L-03 as well as for L-02, L-05, and L-07 in male animals (Figure 4). In female animals, sub-clusters of higher similarity could be observed for indene, naphthalene, the BASF LOA streams, L-03, L-06, and L-11 as well as for L-01, L-02, L-04, L-07, L-08, and L-09 (Figure 5).

 

In order to further investigate potential sub-clusters within the groups of test substances, hierarchical clustering (HCA) as well as principal component analysis (PCA) were performed. In both sexes, indene and naphthalene generally cluster with L-06 and L-11 (Figure 8 – Figure 12, Figure 15, Figure 16). However, using HCA, another sub-cluster including the BASF LOA streams, L-03 and L-11 becomes visible. In female animals, the cluster also includes xylene, the BASF LOA streams and L-03 (HCA and Treatment Correlation (TC)-based clustering, Figure 7 and Figure 9). Another cluster forms with dicyclopentadiene-rich streams (i.e., L-04, L-08 and L-09 in both sexes), L-10 as well as xylene with lower consistency. However, the picture is more scattered between the sexes as can be seen by different other streams falling into these clusters for male and female animals, respectively. It has to be noted that dicyclopentadiene showed a low metabolomic response in female animals, which can explain that this marker compound does not cluster with the respective streams. In male animals, a further cluster can be identified containing L-02, L-05, and L-07. Using different clustering approaches, further compounds (L-01, xylene) fall into this cluster. A summary of the clustering results is given in Figure 28.

 

The toxicity data show qualitatively relatively homogenous effects among all compounds tested (marker compounds, LOA-streams as well as BASF LOA streams). Liver, thyroid, red blood cells, and kidney in males were identified as target organs based on clinical pathology and histopathology investigations. Also, clinical effects were rather comparable for the different compounds. Based on these classical read-outs, no sub-clusters were identifiable in the data set. The effects observed in the classical parameters are supported by the metabolome data. The evaluation of the toxicity patterns in the data base MetaMap®Tox as well as the PCA covering a broader range of different toxicities confirm the limited set of target organ effects. Furthermore, the classical parameters as well as metabolome data suggest the absence of certain toxicities, such as direct thyroid toxicity or other endocrine effects for these streams.

 

Using the metabolome data, sub-clusters of marker compounds, LOA Cat L streams as well as BASF LOA streams could be observed within the limited space of toxicity based on correlation analysis, bootstrapped PCA, and hierarchical clustering (3 clusters in males, 2 clusters in females).

 

The data further imply that toxicity and metabolome effects are partly driven by the marker compounds (based on the 9 compounds tested). However, the marker compounds cannot explain the complete toxicity of the different LOA-streams. Consequently, the LOA-stream toxicity and metabolome effects are not identical to an individual marker substance.

 

Justification for classification or non-classification

There are sufficient data available to conclude that streams within this class which contain less than 1% benzene and less than 10% toluene do not require classification for this endpoint.

Streams which contain ≥1% but less than 10% benzene should be classified as Cat 2, H373 according to Reg (EC) 1272/2008; streams containing ≥10% benzene should be classified as Cat 1, H372 according to Reg (EC) 1272/2008.

Streams which contain ≥10% toluene should be classified as Cat 2, H373 according to Reg (EC) 1272/2008.